Lipid-type substrate GPX activity detection kit, detection method and application

Abstract

The invention provides a lipid-type substrate GPX activity detection kit, a detection method and application, the detection kit comprises the following reagents: AEA-M, dodecyl glucopyranoside, cholesteryl hemisuccinate trihydroxymethyl aminomethane salt, type 1 lipoxygenase and 2, 3-naphthyl diacetal, the 2, 5-anhydro-1-deoxy-D-mannitol is synthesized by an enzymic method. The method is suitable for detecting the enzyme activity of the lipid-type substrate GPX, a reagent reaction system is high in turbidity resistance, the substrate is good in solubility and low-temperature stability, and the method is suitable for samples such as cells and high-fat-content tissue.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a lipid-based substrate GPX activity detection kit, a detection method and an application thereof. Background Art

[0002] Glutathione peroxidase (GPX) is a biological antioxidant enzyme that eliminates substances such as oxygen free radicals generated through enzymatic reactions in vivo and maintains a reduced environment. Currently, it is found that there are 8 types of GPX in glutathione peroxidase, and different types of GPX have inconsistent catalytic activities towards different oxidative substrates. In the study of ferroptosis, iron ions induce lipid peroxidation, leading to the rupture of cell membranes and further causing cell death. Glutathione peroxidase 4, which is sensitive to lipid peroxidation substrates, can eliminate oxidized lipids, thereby inhibiting cell death. Therefore, it is of great significance to study the enzymatic activity of glutathione peroxidase sensitive to its lipid-based substrates.

[0003] GPX catalyzes the generation of oxidized glutathione from reduced glutathione and an oxidative substrate. Currently, the general substrate of GPX enzyme activity detection products sold on the market is an organic hydroxy peroxide, and glutathione reductase is used for enzyme-coupled reactions to catalyze the reaction of reduced coenzyme NADH. The size of GPX enzyme activity is reflected by detecting the decreasing rate of the OD value at a wavelength of 340 nm. The enzyme activity measured by this method is the total enzyme activity of glutathione peroxidase and cannot reflect the ability of GPX to eliminate lipid oxides. The oxidative substrate and coenzyme are not easily preserved at 2 - 8°C and there is natural degradation. Therefore, there is an urgent need for a lipid-based substrate GPX enzyme activity detection product with strong specificity, high detection sensitivity and good stability. Summary of the Invention

[0004] In view of this, the present invention provides a lipid-based substrate GPX activity detection kit, a detection method and an application thereof. The kit has few components, only 4 components, is easy to operate and has few detection steps; the reaction system has no lipid turbidity, the substrate solubility is good; the reagent has good stability, can be detected at room temperature for a long time, and has high sensitivity and can be detected with cell samples.

[0005] AEA-M is a fatty acid substrate that can be oxidized by type 1 lipoxygenase to generate oxidized AEA-M, which can be reduced by glutathione peroxidase. 2,3-Naphthalenedicarboxaldehyde can react with reduced glutathione to generate a fluorescent substance, and this fluorescent substance can be detected at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Dodecylpyranoside and cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt can promote the dissolution of the substrate AEA-M and improve the clarity of the reaction system due to their surfactant effects in the reaction system.

[0006] The technical solution of the present invention is realized as follows:

[0007] In a first aspect, the present invention provides a lipid-based substrate GPX activity detection kit, which includes a substrate solution containing AEA-M, an enzymatic solution, a fluorescent matrix solution, and a standard solution;

[0008] The substrate solution includes AEA-M, the enzymatic solution includes lipoxygenase type 1 and reduced glutathione, the fluorescent matrix solution includes 2,3-naphthalenedicarboxaldehyde, and the structural formula of AEA-M is:

[0009] Based on the above solution, the preparation method of AEA-M includes the following steps. Preferably, weigh 0.304 g of arachidonic acid and 0.326 g of 1-amino-2,5-anhydro-1-deoxy-D-mannitol and add them to a 10 mL round-bottom flask, add 4 mL of n-hexane, add 5 mg of lipase, and react at 40 °C for 6 h under nitrogen protection. After the reaction is completed, wash the reaction solution with pure water 3-4 times, and rotary evaporate to remove the solvent to obtain yellow oily AEA-M.

[0010] Based on the above solution, preferably, the substrate solution further contains dodecylpyranoside, cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt, potassium azide, and Triton X-100.

[0011] Based on the above solution, preferably, in the substrate solution, the concentration of dodecylpyranoside is 1.25 wt% - 3.5 wt%, the concentration of cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt is 0.7 wt% - 3.6 wt%, the concentration of potassium azide is 1 - 5 mmol / L, the concentration of Triton X-100 is 0.5 wt% - 2.5 wt%, the concentration of AEA-M is 2 - 4 mmol / L, and the solvent is a Tris-HCl buffer solution with a pH of 7 - 9 and a concentration of 20 - 150 mmol / L. Further preferably, the concentration of dodecylpyranoside is 2.5 wt%, the concentration of cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt is 2.1 wt%, the concentration of potassium azide is 2.5 mmol / L, the concentration of Triton X-100 is 0.8 wt%, the concentration of AEA-M is 3 mmol / L, and the solvent is a Tris-HCl buffer solution with a pH of 8.4 and a concentration of 100 mmol / L.

[0012] Based on the above solution, preferably, the enzymatic solution further contains trehalose, bovine serum albumin, and glycerol;

[0013] Based on the above - mentioned scheme, preferably, in the enzymatic solution, the concentration of trehalose is 1.6 wt% - 6.0 wt%, the concentration of bovine serum albumin is 0.5 wt% - 1 wt%, the concentration of glycerol is 0.5 wt% - 2.5 wt%, the concentration of type 1 lipoxygenase is 50 - 250 U / mL, the concentration of reduced glutathione is 2 - 5 mmol / L, and the solvent is Tris - HCl buffer solution with pH 7 - 9 and 20 - 150 mmol / L. Further preferably, the concentration of trehalose is 4.3 wt%, the concentration of bovine serum albumin is 0.6 wt%, the concentration of glycerol is 1.2 wt%, the concentration of type 1 lipoxygenase is 150 U / mL, the concentration of reduced glutathione is 2.5 mmol / L, and the solvent is Tris - HCl buffer solution with pH 8.4 and 100 mmol / L.

[0014] Based on the above - mentioned scheme, the fluorescent substrate solution further contains ammonium acetate. Preferably, the concentration of ammonium acetate is 0.01 wt% - 0.03 wt%, and the concentration of 2,3 - naphthalenedicarboxaldehyde is 20 - 30 mmol / L. Further preferably, the concentration of ammonium acetate is 0.02 wt%, and the concentration of 2,3 - naphthalenedicarboxaldehyde is 25 mmol / L.

[0015] In the second aspect, the present invention provides a detection method for the kit as described above, comprising the following steps:

[0016] S1, processing of animal tissue samples or collected cell samples. Preferably, homogenize according to the ratio of tissue sample mass (g): normal saline volume (mL)=1:9 (for example, for 0.05 g of tissue sample, add 0.45 mL of normal saline), centrifuge at 10000×g for 10 min at 4°C, and take the supernatant for testing; according to the ratio of 1×10 6 cells (g): normal saline volume (mL)=1:0.2 mL (for example, for 1×10 6 cells, add 0.2 mL of normal saline), centrifuge at 10000×g for 10 min at 4°C, and take the supernatant for testing;

[0017] S2, set blank wells, standard wells, and measurement wells on the fluorescence microplate;

[0018] S3, add double - distilled water to the blank wells, add standard products with different concentrations to the standard wells, and add the supernatant in step S1 to the measurement wells;

[0019] S4, add an equal volume of substrate solution and enzymatic solution to the measurement wells in step S3, and incubate for reaction. Preferably, the incubation reaction temperature is 37°C and the time is 20 min;

[0020] S5. Add equal volumes of the fluorescent substrate solution to the standard wells, blank wells, and the measurement wells in step S4 in sequence, and incubate for the reaction. Preferably, the incubation temperature is 37 °C and the time is 10 min.

[0021] S6. Set the excitation wavelength of the fluorescence microplate reader to 350 nm and the emission wavelength to 450 nm, detect the fluorescence values of the wells on the fluorescence plate, and fit the standard curve. Preferably, fit the standard curve according to the absolute fluorescence values of the standard wells and the concentrations of the standard products.

[0022] When the sample to be tested is a cell or tissue sample, use the following formula to calculate the enzyme activity:

[0023]

[0024] Wherein, F1: the fluorescence value of the measurement well; F2: the fluorescence value of the blank well; f: the conversion coefficient between the standard well system and the measurement well system; T: the fixed incubation time; Cpr: the protein concentration (g prot / L) when the sample to be tested is added to the detection system.

[0025] The definition of U is: in the reaction system, the amount of enzyme required for 1 μmol of the substrate reduced glutathione to be reduced per minute per gram of protein at a specific temperature is one activity unit.

[0026] In the third aspect, the present invention provides the application of the above-mentioned kit in detecting the GPX enzyme activity in a sample.

[0027] The lipid-based substrate GPX activity detection kit, detection method, and application of the present invention have the following beneficial effects compared with the prior art:

[0028] (1) The kit of the present invention has high detection sensitivity and is applicable to detecting cell samples; the kit has fewer reagent components and is convenient to operate.

[0029] The present invention uses the reduced fatty substrate AEA-M, which can specifically detect the GPX enzyme activity of the fatty substrate, and has strong thermal stability, good water solubility, and can maintain a long reaction activity at room temperature.

[0030] (2) The substrate solution dodecylpyranoside and cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt used in the present invention promote the dissolution of the substrate AEA-M due to their surfactant effects, ensuring the clarity of the system. Description of the Drawings

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0032] Figure 1 This is the schematic diagram of the GPX enzyme activity detection of the present invention;

[0033] Figure 2 This is the fitting curve diagram of the standard product of the present invention;

[0034] Figure 3 This is the comparison diagram of the results of the detection method of the reference document of the present invention and this detection method;

[0035] Figure 4 This is the comparison diagram of the reagent stability results of the present invention;

[0036] Figure 5 This is the optimization diagram of the turbidity situation of the system of the present invention. Specific embodiments

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0038] Example 1: Synthesis of lipid-type substrate AEA-M

[0039] Weigh 0.304 g of arachidonic acid and 0.326 g of 1-amino-2,5-anhydro-1-deoxy-D-mannitol and add them to a 10 mL round-bottom flask. Add 4 mL of n-hexane and 5 mg of lipase. React at 40 °C for 6 h under nitrogen protection. After the reaction is completed, wash the reaction solution with pure water 3 - 4 times and remove the solvent by rotary evaporation under vacuum. A yellow oily product is obtained with a yield of 65%. The purity is determined by 1H NMR (400 MHz, Chloroform-d). δ 8.0 (1H, s), 5.85 (1H, d), 5.51 - 5.41 (8H, m), 5.41 (2H, m), 4.71 (2H, m), 3.50 (2H, m), 2.8 (6H, dd, J1 = 8.5 Hz, J2 = 2.9 Hz), 2.34 (2H, t, J = 7.5 Hz), 2.16 (4H, m), 1.79 (2H, m), 1.29 (6H, m), 0.88 (3H, m). 13C NMR (101 MHz, Chloroform-d) δ 172.5, 130.3, 128.8, 127.7, 127.4, 102.4, 84.1, 76.4, 69.5, 40.1, 37.7, 36.6, 33.8, 32.8, 31.9, 29.6, 25.5, 21.8, 14.1.

[0040] Example 2: Preparation of a lipid-based GPX enzyme activity detection kit

[0041] A lipid-based GPX enzyme activity detection kit contains the following reagents: substrate solution, enzyme reaction solution, fluorescent substrate solution, standard solution.

[0042] Preparation of the substrate solution: AEA-M 3 mmol / L, dodecylpyranoside 2.5 wt%, cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt 2.1 wt%, potassium azide 2.5 mmol / L, Triton X-100 0.8 wt%, and the solvent is 100 mmol / L Tris-HCl buffer solution with pH 8.4.

[0043] Preparation of the enzyme reaction solution: Lipoxygenase type 1 150 U / mL, trehalose 4.3 wt%, bovine serum albumin 0.6 wt%, glycerol 1.2 wt%, reduced glutathione 2.5 mmol / L, and the solvent is 100 mmol / L Tris-HCl buffer solution with pH 8.4.

[0044] Preparation of the fluorescent substrate solution: 2,3-naphthalenedicarboxaldehyde 25 mmol / L, ammonium acetate wt 0.02%;

[0045] Preparation of the standard solution: Reduced glutathione 1 mmol / L.

[0046] Example 3: Preparation of a lipid-based GPX enzyme activity detection kit

[0047] A lipid-based GPX enzyme activity detection kit, comprising the following reagents: substrate solution, enzyme reaction solution, fluorescent matrix solution, standard solution.

[0048] Preparation of substrate solution: AEA-M 2 mmol / L, dodecylpyranoside 1.25 wt%, cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt 0.7 wt%, potassium azide 1 mmol / L, Triton X-100 0.5 wt%, and the solvent is Tris-HCl buffer solution with pH 7 and 20 mmol / L.

[0049] Preparation of enzyme reaction solution: Lipoxygenase type 1 50 U / mL, trehalose 1.6 wt%, bovine serum albumin 0.5 wt%, glycerol 0.5 wt%, reduced glutathione 2 mmol / L, and the solvent is Tris-HCl buffer solution with pH 7 and 20 mmol / L.

[0050] Preparation of fluorescent matrix solution: 2,3-naphthalenedicarboxaldehyde 20 mmol / L, ammonium acetate 0.01 wt%;

[0051] Preparation of standard solution: Reduced glutathione 1 mmol / L.

[0052] Example 4: Preparation of lipid-based GPX enzyme activity detection kit

[0053] A lipid-based GPX enzyme activity detection kit, comprising the following reagents: substrate solution, enzyme reaction solution, fluorescent matrix solution, standard solution.

[0054] Preparation of substrate solution: AEA-M 4 mmol / L, dodecylpyranoside 3.5 wt%, cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt 3.6 wt%, potassium azide 5 mmol / L, Triton X-100 2.5 wt%, and the solvent is Tris-HCl buffer solution with pH 9 and 150 mmol / L.

[0055] Preparation of enzyme reaction solution: Lipoxygenase type 1 250 U / mL, trehalose 6.0 wt%, bovine serum albumin 1.0 wt%, glycerol 2.5 wt%, reduced glutathione 5 mmol / L, and the solvent is Tris-HCl buffer solution with pH 9 and 150 mmol / L.

[0056] Preparation of fluorescent matrix solution: 2,3-naphthalenedicarboxaldehyde 30 mmol / L, ammonium acetate 0.03 wt%;

[0057] Preparation of standard solution: Reduced glutathione 1 mmol / L.

[0058] Example 5: Detection method of lipid-based GPX enzyme activity detection kit

[0059] Detection method of a lipid-based GPX enzyme activity detection kit, the specific steps are as follows:

[0060] (1) Experimental process

[0061] 1. Treatment of animal tissue samples or collected cell samples:

[0062] Homogenize according to the ratio of tissue sample mass (g): normal saline volume (mL) = 1:9 (e.g., for 0.05 g of tissue sample, add 0.45 mL of normal saline, centrifuge at 10000×g for 10 min at 4°C, and take the supernatant for testing. For 1×10 6 cells (g): normal saline volume (mL) = 1:0.2 mL (e.g., for 1×10 6 cells, add 0.2 mL of normal saline), centrifuge at 10000×g for 10 min at 4°C, and take the supernatant for testing.

[0063] 2. Set blank wells, standard wells, and measurement wells on the fluorescence microplate, and set the positions of each well according to Table 1:

[0064] Table 1 Well position setting table

[0065] 1 2 3 4 5 6 7 8 9 10 11 12 A S1 S9 S17 S25 S33 S41 S49 S57 S65 S73 S81 S89 B S2 S10 S18 S26 S34 S42 S50 S58 S66 S74 S82 S90 C S3 S11 S19 S27 S35 S43 S51 S59 S67 S75 S83 S91 D S4 S12 S20 S28 S36 S44 S52 S60 S68 S76 S84 S92 E S5 S13 S21 S29 S37 S45 S53 S61 S69 S77 S85 S93 F S6 S14 S22 S30 S38 S46 S54 S62 S70 S78 S86 S94 G S7 S15 S23 S31 S39 S47 S55 S63 S71 S79 S87 S95 H S8 S16 S24 S32 S40 S48 S56 S64 S72 S80 S88 S96

[0066] Among them: S1 - S2: blank wells; S3 - S16: standard wells; S17 - S96: measurement wells, where S17 - S43 correspond to Example 2, S44 - S70 correspond to Example 3, and S71 - S96 correspond to Example 4.

[0067] 3. Add 20 μL of double-distilled water to the blank wells, 120 μL of standard products with different concentrations to the standard wells, and 20 μL of the supernatant of the sample homogenate to the measurement wells.

[0068] 4. Add 50 μL of substrate solution and 50 μL of enzyme reaction solution to the measurement wells and blank wells in step 3 in sequence, and incubate at 37°C for 20 min.

[0069] 5. Add 100 μL of fluorescence substrate solution to the standard wells, blank wells, and measurement wells in step 4, and incubate at 37°C for 10 min.

[0070] 6. Set the excitation wavelength of the fluorescence microplate reader at 350 nm and the emission wavelength at 450 nm, and detect the fluorescence values of the wells on the fluorescence plate.

[0071] Example 6:

[0072] Using the kit in Examples 2-4 and the detection method in Example 5, detection instrument: the multi-functional microplate reader FlexStation 3 from Molecular Devices, USA; six samples were selected: 10% rat kidney tissue homogenate, 10% rat liver tissue homogenate, 10% rat brain tissue homogenate, 10% mouse kidney tissue homogenate, 293T cells, Hela cells, and recombinant protein GPX-4 for detection. Each standard was detected twice and each sample was detected three times. The results are as follows:

[0073] The fluorescence values of the standard detections are shown in Table 2:

[0074] Table 2 Fluorescence values of standard detections

[0075]

[0076] The standard fitting curve is as Figure 2 shown.

[0077] When the samples to be detected are cell or tissue samples, the following formula is used to calculate the enzyme activity:

[0078]

[0079] where, F1: fluorescence value of the measurement well, F2: fluorescence value of the blank well, f: conversion coefficient between the standard well system and the measurement well system, T: incubation and fixation time, Cpr: protein concentration (g prot / L) when the sample to be detected is added to the detection system.

[0080] The definition of U is: in the reaction system, the amount of enzyme required for 1 μmol of the substrate reduced glutathione to be reduced per minute per gram of protein at a specific temperature is one activity unit.

[0081] The fluorescence values and enzyme activities of the sample detections are shown in Table 3:

[0082] Table 3 Fluorescence values and enzyme activities of sample detections

[0083]

[0084] It can be seen from the above experimental results that the AEA-M detection method is applicable to various animal tissue and cell samples, and has diverse application scenarios.

[0085] Comparative Example 1

[0086] Using the glutathione peroxidase enzyme activity detection method described in the literature (International Dairy Journal 10.5-6(2000):347-351) to detect common laboratory samples. This method was operated with reference to Example 1 and Example 2. The detection results are as Figure 3 shown:

[0087] The detection effect of the tissue sample is consistent with that of the commercially available product. In the detection results of the AEA method, the GPX-4 detection enzyme activity of the fatty substrate is better, and the GPX1 detection enzyme activity of the non-lipid type is lower; the value can still be measured when detecting cell samples.

[0088] Comparative Example 2

[0089] Referring to the detection method in the literature of Comparative Example 1, the reagent was stored at 2-8 °C for an extended time of 2 months, and the enzyme activity of the liver tissue sample stored at -80 °C was detected. The detection results are as Figure 4 shown:

[0090] For the AEA-M detection method, the reagent has better thermal stability and can maintain longer reaction activity at low temperature.

[0091] Comparative Example 3

[0092] After adding dodecyl glucopyranoside and cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt to the detection system, the dissolution of the lipid substrate can be significantly improved, ensuring the clarity and stability of the system, and improving the anti-interference ability of the reagent. As Figure 5 The two holes in the left column are added, Figure 5 The two holes in the right column are not added.

[0093] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A lipid-based substrate GPX activity detection kit, characterized in that, It includes the following reagents: substrate solution, enzymatic solution, fluorescent matrix solution and standard solution; The substrate solution includes AEA-M, the enzymatic solution includes lipoxygenase type 1 and reduced glutathione, and the fluorescent matrix solution includes 2,3-naphthyldialdehyde. The structural formula of AEA-M is:

2. The kit according to claim 1, wherein The preparation method of AEA-M includes the following steps: Dissolve arachidonic acid and 1-amino-2,5-anhydro-1-deoxy-D-mannitol in n-hexane, add lipase, and react at 35-45 °C for 5-7 h under nitrogen protection. After the reaction is completed, wash with pure water 3-4 times, and remove the solvent by rotary evaporation to obtain yellow oily AEA-M.

3. The kit according to claim 2, characterized in that, The mass ratio of arachidonic acid: 1-amino-2,5-anhydro-1-deoxy-D-mannitol: lipase is 300-310:320-330:

5.

4. The kit according to claim 1, characterized in that, The substrate solution also contains dodecylpyranoside, cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt, potassium azide and Triton X-100.

5. The kit according to claim 4, characterized in that In the substrate solution, the concentration of dodecylpyranoside is 1.25 wt%-3.5 wt%, the concentration of cholesteryl hemisuccinate tris(hydroxymethyl)aminomethane salt is 0.7 wt%-3.6 wt%, the concentration of potassium azide is 1-5 mmol / L, the concentration of Triton X-100 is 0.5 wt%-2.5 wt%, the concentration of AEA-M is 2-4 mmol / L, and the solvent is Tris-HCl buffer solution with pH 7-9 and 20-150 mmol / L.

6. The kit according to claim 1, wherein, The enzymatic solution also contains trehalose, bovine serum albumin and glycerol.

7. The kit according to claim 6, wherein, In the enzymatic solution, the concentration of trehalose is 1.6 wt%-6.0 wt%, the concentration of bovine serum albumin is 0.5 wt%-1 wt%, the concentration of glycerol is 0.5 wt%-2.5 wt%, the concentration of lipoxygenase type 1 is 50-250 U / mL, the concentration of reduced glutathione is 2-5 mmol / L, and the solvent is Tris-HCl buffer solution with pH 7-9 and 20-150 mmol / L.

8. The kit according to claim 1, characterized in that The fluorescent matrix solution also contains ammonium acetate, and the concentration of ammonium acetate is 0.01 wt%-0.03 wt%, and the concentration of 2,3-naphthyldialdehyde is 20-30 mmol / L.

9. A detection method for the kit according to claim 1, characterized in that, It includes the following steps: S1, Homogenize the animal tissue sample or the collected cell sample and then centrifuge to obtain the supernatant; S2, Set blank wells, standard wells and assay wells on the fluorescence microplate; S3, Add double-distilled water to the blank wells, add standard products with different concentrations to the standard wells, and add the supernatant of step S1 to the assay wells; S4, Add an equal volume of substrate solution and enzymatic solution to the assay wells in step S3, and incubate for reaction; S5, Add an equal volume of fluorescent matrix solution to the standard wells, blank wells and the assay wells in step S4 in sequence, and incubate for reaction; S6, Set the excitation wavelength of the fluorescence microplate reader to 350 nm and the emission wavelength to 450 nm, detect the fluorescence values of the microplate wells, fit the standard curve, and calculate the enzyme activity.

10. Use of a kit according to any one of claims 1-8 for detecting GPX enzyme activity in a sample.

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