A method for rapid detection of neutrophil extracellular traps

Abstract

The present invention relates to the field of medical testing technology, and in particular to a method for rapidly detecting neutrophil extracellular traps. The method comprises the following steps: (1) taking a human whole blood sample, adding a hemolytic agent, and then adding a mixed fluorescent antibody staining solution, shaking and mixing, and incubating at room temperature; (2) centrifuging after incubation in step (1), discarding the supernatant, washing with a washing solution, and then centrifuging, discarding the supernatant; (3) adding a washing solution to resuspend the cells; (4) using a flow cytometer for detection. The detection method has good stability, strong repeatability, and high fluorescence intensity, and can be used for auxiliary diagnosis of diseases.

Description

Technical Field

[0001] The invention relates to the technical field of medical testing, and in particular to a method for rapidly detecting neutrophil extracellular traps. Background Art

[0002] Neutrophils play a very important role in the nonspecific cellular immune system of the blood. They are at the front line of the body's defense against microbial pathogens, especially in the invasion of purulent bacteria. When inflammation occurs, they are attracted to the site of inflammation by chemotactic substances. Because they obtain energy through glycolysis, they can still survive in the hypoxic conditions of swelling and poor blood flow. Here they form cytotoxicity to destroy the cell membranes of bacteria and nearby tissues. Since neutrophils contain a large number of lysosomal enzymes, they can decompose the bacteria and tissue fragments engulfed into the cells. In this way, the invading bacteria are surrounded in a local area and eliminated, preventing the spread of pathogenic microorganisms in the body. When the neutrophils themselves disintegrate, the release of various lysosomal enzymes can dissolve the surrounding tissues to form abscesses. Neutrophils contain many diffusely distributed small light red or light purple unique granules, which contain myeloperoxidase and some other enzymes. Myeloperoxidase is unique to neutrophils, and even in macrophages with strong phagocytic function, there is little or no such enzyme. In cell chemistry, myeloperoxidase is generally used as a marker of neutrophils. After being stimulated by extracellular physical and chemical factors, the immune function of neutrophils is activated, and the pore-punching proteins GSDMD (involved in cell pyroptosis) and MLKL (involved in cell necrosis) will punch holes in the neutrophil membrane, causing calcium ions to flow into the cell; subsequently, peptidyl arginine deiminase 4 (PAD4) will be activated, and the activated PAD4 will citrullinate histones; this change will cause DNA to unwind from histones, the nuclear chromatin will lose its normal shape, and DNA, histones and myeloperoxidase in intracellular vesicles will combine and be released as the cell membrane ruptures, forming a neutrophil extracellular trap. Clinical medical research has shown that neutrophil extracellular traps are related to major medical diseases such as inflammation, sepsis, cancer, myocardial infarction, stroke, and autoimmune diseases. In patients with the above diseases, an increase in the content of neutrophil extracellular traps has been detected. The detection of neutrophil extracellular traps can indicate the pathological state of the disease and provide guidance for disease treatment and prognosis. Although the detection of neutrophil extracellular traps has important clinical value, there is still a lack of methods for detecting neutrophil extracellular traps in clinical specimens. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention proposes a method for rapidly detecting neutrophil extracellular traps.

[0004] The present invention is achieved through the following technical solutions:

[0005] A method for rapidly detecting neutrophil extracellular traps comprises the following steps:

[0006] (1) Take a whole blood sample, add a hemolytic agent, then add a mixed fluorescent antibody staining solution, shake and mix, and incubate at room temperature for 30 min;

[0007] (2) After incubation in step (1), centrifuge at 400 g for 5 min, discard the supernatant, wash three times with washing solution, centrifuge at 400 g for 5 min, and discard the supernatant;

[0008] (3) Add washing buffer to resuspend the cells;

[0009] (4) Detection by flow cytometry.

[0010] Furthermore, in step (1), the volume ratio of the human whole blood sample, the hemolytic agent and the mixed fluorescent antibody staining solution is 1:1:0.1.

[0011] Furthermore, in step (1), the preparation steps of the hemolytic agent are as follows: 8 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L sodium dihydrogen phosphate, and 0.24 g / L potassium dihydrogen phosphate are used to prepare a solution, and the pH value is adjusted to 7.4 with hydrochloric acid to obtain a hemolytic agent.

[0012] Furthermore, in step (1), the method for preparing the mixed fluorescent antibody staining solution comprises the following steps:

[0013] (11) Preparation of modified eight-arm polyethylene glycol:

[0014] (111) A mixed solution of anhydrous ethanol and deionized water (v:v = 2:1) was ultrasonically degassed for 10 min, and 10 kDa eight-arm polyethylene glycol hydroxyl and 3-aminopropyltriethoxysilane (APTES) were mixed and added to the ultrasonically degassed mixed solution of anhydrous ethanol and deionized water. After ultrasonic treatment at 200 W for 20 min under N 2 protection, ammonia water was added to adjust the pH value to 8–9, and the mixture was shaken at 300 rpm and 50 °C for 3 h to obtain a mixed solution.

[0015] (112) Chlorohexane was added to the mixed solution obtained in step (111), the temperature was raised to 80°C, and the mixture was stirred at 120 rpm for 6-8 h. The mixture was allowed to stand at 4°C for 4-6 h, and centrifuged at 10,000 rpm for 15 min at 4°C. The precipitate was collected, and washed repeatedly with acetone, anhydrous ethanol, and deionized water. The mixture was dried in a vacuum oven at 50°C to constant weight to obtain modified eight-arm polyethylene glycol.

[0016] Furthermore, in step (111), the ratio of anhydrous ethanol, eight-arm polyethylene glycol hydroxyl and APTES is 0.5 mL:10 mg:1 mg.

[0017] Furthermore, in step (112), the mass ratio of the chlorohexane to the eight-arm polyethylene glycol hydroxyl group in step (111) is 1:4.

[0018] (12) Preparation of liposomes@modified GO-fluorescent dye:

[0019] (121) Graphene oxide (GO) was prepared by the Hummers method. The graphene oxide solid sample was dispersed in distilled water (1 mg / mL). The solution was intermittently sonicated at 600 W for 2 h (on 1 s, off 2 s) using a cell disruptor to obtain a treated GO solution. The treated GO solution was added with 2 mg / mL EDC solution and sonicated at 570 W for 30 min (on 1.5 s, off 1 s). The modified eight-arm polyethylene glycol obtained in step (112) was added and sonicated at 300 W for 30 min. The solution was stirred at 180 rpm for 12 h and centrifuged at 8000 rpm for 30 min. The precipitate was washed with deionized water and dried in a vacuum at 25 °C to obtain modified GO.

[0020] (122) 1,2-Dimyristoylphosphatidylglycerol (DMPG) and dipalmitoylphosphatidylethanolamine (DPPE) were dissolved in chloroform / methanol solution (V 氯仿 :V 甲醇 =2:1), placed in a round-bottom flask, and evaporated the organic solvent under reduced pressure on a rotary evaporator at 30°C in a water bath to obtain a lipid film on the inner wall of the round-bottom flask, and dried the round-bottom flask containing the lipid film in a vacuum dryer for 16-18 h to completely remove the residual organic solvent, and added ultrapure water to the lipid film, and ultrasonicated at 300 W for 10 min in a 60°C water bath to obtain a hydrated liposome solution, and used a syringe to pass the above hydrated liposome solution twice through a 0.22 μm polycarbonate membrane to obtain a liposome solution;

[0021] (123) A 10 mg / mL dye aqueous solution was mixed with a 1 mg / mL modified GO aqueous solution obtained in step (121), stirred at 100 rpm for 2-3 h to obtain a dye-GO solution, and the dye-GO solution was mixed with the liposome solution obtained in step (122), ultrasonically treated at 200 W for 8 h in a 0 °C ice water bath, and centrifuged at 3000 rpm for 15 min. The supernatant was centrifuged 3-5 times through an ultrafiltration tube with a molecular weight cutoff of 100 kDa to obtain liposomes@modified GO-fluorescent dye.

[0022] Furthermore, in step (121), the volume ratio of the treated GO solution to the 2 mg / mL EDC solution is 5:1.

[0023] Furthermore, in step (121), the ratio of the modified eight-arm polyethylene glycol to the treated GO solution is 15 mg:4 mL.

[0024] Furthermore, in step (122), the molar ratio of DMPG to DPPE is 4:1.

[0025] Furthermore, in step (122), the dosage ratio of DMPG to chloroform / methanol solution is 0.8 μmol:1 mL.

[0026] Furthermore, in step (122), the volume ratio of the ultrapure water to the chloroform / methanol solution is 4:3.

[0027] Furthermore, in step (123), the volume ratio of the dye aqueous solution, the modified GO aqueous solution and the liposome solution is 2:5:20.

[0028] Furthermore, in step (123), the dye is one of FITC, PreCP, APC and PE, which are commonly used dyes in flow cytometry, and the labeling of the antibody and the fluorescent dye can be arbitrarily combined.

[0029] (13) Preparation of liposome fluorescently labeled antibodies:

[0030] (131) S-SMCC was dissolved in sterile water to prepare a 10 mg / mL S-SMCC solution. The liposome@modified GO-fluorescent dye obtained in step (123) was dispersed in ultrapure water at an amount of 15 mg / mL to obtain a liposome@modified GO-fluorescent dye dispersion. The S-SMCC solution was added to the dispersion and reacted at 25 °C for 1-2 h.

[0031] (132) The solution after the reaction in step (131) was transferred to an ultrafiltration centrifuge tube, and PBS (containing 0.25 mM EDTA) buffer was added. The solution was centrifuged at 12000 g for 5 min and the filtrate was removed. PBS (containing 0.25 mM EDTA) buffer was added and mixed and centrifuged. This operation was repeated 5 times for desalting. The desalted product was collected and fixed to volume with pure water to obtain a liposome@modified GO-fluorescent dye-S-SMCC solution.

[0032] (133) The MPO antibody was diluted to 2 mg / mL with PBS to obtain an antibody solution. Sulfo-LC-SPDP was dissolved in PBS to a concentration of 0.05 mg / mL. Sulfo-LC-SPDP solution was added to the antibody solution to make the final concentration of Sulfo-LC-SPDP 1.5 μg / mL. PBS was added to the antibody concentration to 0.5 mg / mL. The mixture was reacted at 25 °C for 1 h. The mixture was transferred to an ultrafiltration centrifuge tube. PBS (containing 0.25 mM EDTA) buffer was added. The mixture was centrifuged at 12,000 g for 5 min and the filtrate was removed. PBS (containing 0.25 mM EDTA) buffer was added and mixed and centrifuged. This operation was repeated 5 times. The antibody solution was fixed to volume with pure water.

[0033] (134) Dissolve TCEP in PBS to a final concentration of 0.05 mg / mL, add the TCEP solution to the antibody solution obtained in step (133) to make a final TCEP concentration of 1 μg / mL, react at 25°C in the dark for 1 h, transfer to an ultrafiltration centrifuge tube, add PBS (containing 0.25 mM EDTA) buffer, centrifuge at 12,000 g for 5 min, remove the filtrate, add PBS (containing 0.25 mM EDTA) buffer, mix and centrifuge, repeat this operation 5 times, fix the volume, and obtain McAb-SH intermediate solution;

[0034] (135) The liposome@modified GO-fluorescent dye-S-SMCC solution and the McAb-SH intermediate solution obtained in step (132) and step (134) were mixed evenly and reacted in the dark at 25 °C for 3 h. After the reaction was completed, 0.1 mg / mL PBS (containing 1% BSA, 0.3 mg / mL purslane extract, and 0.2 mg / mL neem extract) solution was added to the reaction system to make the final antibody concentration 0.1 mg / mL to obtain MPO liposome fluorescently labeled antibody solution.

[0035] Furthermore, in step (131), the volume ratio of the liposome@modified GO-fluorescent dye dispersion to the S-SMCC solution is 10:1.

[0036] Furthermore, in step (135), the volume ratio of the liposome@modified GO-fluorescent dye-S-SMCC solution to the McAb-SH intermediate solution is 4-7:5.

[0037] Furthermore, in step (135), the preparation steps of the purslane and neem extract are as follows: wash the purslane and neem, dry them at 80°C, crush them through a 100-mesh sieve, add them into 10 times the mass volume of deionized water, heat them in a water bath at 70-80°C, stir them at 400-500 rpm for 1-2 h, centrifuge them at 8000 rpm for 5 min, filter them, continue the above heating and stirring for the filtrate for 6 h, centrifuge them at 8000 rpm for 5 min, filter them through a 0.22 μm micromembrane, and freeze-dry them at -20°C for 12 h to obtain the extract.

[0038] (14) Preparing a mixed fluorescent antibody staining solution: Prepare MPO, CitH3, CD66c and CD45 liposome fluorescently labeled antibody solutions by the method of step (13), and mix them evenly to form a mixed fluorescent antibody staining solution.

[0039] Furthermore, in step (14), the volume ratio of the MPO, CitH3, CD66c and CD45 liposome fluorescently labeled antibody solution is 0.8-1.5:0.6-1.2:0.5-1:0-1.

[0040] Furthermore, in step (2), the washing solution is prepared by dissolving BSA, Proclin 300 and tween 20 in PBS buffer at mass fractions of 1%, 0.1% and 1%.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The invention provides a method for rapidly detecting neutrophil extracellular trapping nets. The method improves the performance of a mixed fluorescent antibody staining solution, improves the stability of a fluorescent dye by improving graphene oxide and liposomes, and thus enhances the detection capability of neutrophils. In the mixed fluorescent antibody staining solution of the invention, graphene oxide is used as a matrix material for loading fluorescent dyes. Graphene oxide (GO) has an ultra-large specific surface area and contains a large number of active groups, and can load fluorescent dyes, thereby enhancing stability and reducing leakage. However, fluorescent dyes are easily quenched when directly connected to GO. Graphene oxide is modified with modified eight-arm polyethylene glycol, and carboxyl groups on graphene oxide are activated with EDC, and graphene oxide is connected to the modified eight-arm polyethylene glycol through a covalent bond. The modified GO is combined with the fluorescent dye through the modified eight-arm polyethylene glycol, thereby avoiding fluorescent dye quenching caused by directly loading the fluorescent dye, and realizing stable fluorescent labeling. The modified eight-arm polyethylene glycol prepared by the invention is aminoated by introducing an APTES group, and the introduction of the APTES group can improve stability. , reducing fluorescence quenching, improving the interfacial binding performance of the eight-arm polyethylene glycol hydroxyl group grafted with the silane coupling agent, retaining the amino end of APTES after combining with the eight-arm polyethylene glycol hydroxyl group, and the amino group can act as a nucleophilic agent to attack the halogenated carbon atom on the halogenated hydrocarbon, causing a quaternary ammonium salt reaction, so that the reaction product has a positive charge, and is connected to the negatively charged or uncharged fluorescent dye through electrostatic interaction, thereby loading the fluorescent dye on the modified GO, and at the same time, the modified eight-arm polyethylene glycol hydroxyl group introduces Long-chain hydrophobic alkyl groups improve hydrophobicity; the present invention uses 1,2-dimyristoylphosphatidylglycerol (DMPG) and dipalmitoylphosphatidylethanolamine (DPPE) as liposomes. DMPG can make liposomes form and carry negative charges. DPPE has amino groups in its molecular structure and can be coupled with antibodies. The phospholipid molecules are hydrophobic, with the hydrophobic tail facing the modified GO and the hydrophilic head facing the outside. Because the surface carries a negative charge, the dispersibility is improved by electrostatic force to avoid the aggregation of fluorescent dyes. The fluorescent dye is encapsulated in the liposome. Due to the protection of the outer layer of the liposome, the fluorescent substance is not easy to be decomposed, exists relatively stably, is stable to light, is not easy to quench, avoids the leakage of the fluorescent dye, and further improves the detection accuracy. The fluorescent liposomes prepared by the present invention have good dispersibility, uniform particle size, and strong fluorescence. The present invention adds purslane and neem extracts, contains antioxidant active ingredients, can improve stability, reduce antibody denaturation in a complex reaction environment, stabilize the antibody structure, ensure that the antibody maintains the specific binding ability with the antigen, and maintains the accuracy of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 This is a scanning electron micrograph of the liposome@modified GO-fluorescent dye described in Example 1 of the present invention;

[0045] Figure 2 This is the flow cytometric diagram of Experimental Example 1 of the present invention, wherein the left diagram is of a normal person and the right diagram is of an inflammatory patient;

[0046] Figure 3 CV graphs of Examples 1-3 and Comparative Examples 1-3 of the present invention;

[0047] Figure 4 CV graphs after storage of Examples 1-3 and Comparative Examples 1-3 of the present invention;

[0048] Figure 5 It is the rate of decrease of fluorescence intensity before and after storage described in Examples 1-3 of the present invention and Comparative Examples 1-3. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0050] Example 1: This example provides a method for rapidly detecting neutrophil extracellular traps, comprising the following steps:

[0051] (1) Accurately weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium dihydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate and dissolve them in distilled water. Adjust the pH value to 7.4 with hydrochloric acid and add distilled water to make up to 1000 mL to obtain a hemolytic agent. Take 1000 μL of human whole blood sample, add 1000 μL of the hemolytic agent, and then add 100 μL of the mixed fluorescent antibody staining solution. After vortexing and mixing, incubate at room temperature for 30 min.

[0052] In step (1), the method for preparing the mixed fluorescent antibody staining solution comprises the following steps:

[0053] (11) Preparation of modified eight-arm polyethylene glycol:

[0054] (111) A mixed solution of 0.5 mL of anhydrous ethanol and deionized water (v:v = 2:1) was ultrasonically degassed for 10 min, and 10 mg of 10 kDa eight-arm polyethylene glycol hydroxy and 1 mg of 3-aminopropyltriethoxysilane (APTES) were mixed and added to the ultrasonically degassed mixed solution of anhydrous ethanol and deionized water. The mixture was ultrasonically treated at 200 W for 20 min under N 2 protection, and then ammonia was added to adjust the pH value to 9. The mixture was shaken at 300 rpm and 50 °C for 3 h to obtain a mixed solution.

[0055] (112) Add 2.5 mg of chlorohexane to the mixed solution obtained in step (111), heat to 80°C, stir at 120 rpm for 8 h, stand at 4°C for 6 h, centrifuge at 10,000 rpm for 15 min at 4°C, collect the precipitate, wash repeatedly with acetone, anhydrous ethanol, and deionized water, and dry in a vacuum drying oven at 50°C to constant weight to obtain modified eight-arm polyethylene glycol;

[0056] (12) Preparation of liposomes@modified GO-fluorescent dye:

[0057] (121) Graphene oxide (GO) was prepared by the Hummers method. The graphene oxide solid sample was dispersed in distilled water (1 mg / mL). The GO solution was obtained by intermittent ultrasonication at 600 W for 2 h (on 1 s, off 2 s) using a cell crusher. 80 mL of the treated GO solution was added to 16 mL of 2 mg / mL EDC solution. The solution was ultrasonicated at 570 W for 30 min (on 1.5 s, off 1 s). 300 mg of the modified eight-arm polyethylene glycol obtained in step (112) was added. The solution was ultrasonicated at 300 W for 30 min, stirred at 180 rpm for 12 h, and centrifuged at 8000 rpm for 30 min. The precipitate was washed with deionized water and dried in a vacuum at 25 °C to obtain the modified GO.

[0058] (122) 1,2-Dimyristoylphosphatidylglycerol (DMPG) and dipalmitoylphosphatidylethanolamine (DPPE) were dissolved in 3 mL of chloroform / methanol solution (V 2.5) at a molar ratio of 4:1 (total amount of substance was 3 μmol). 氯仿 :V 甲醇 =2:1), placed in a round-bottom flask, evaporated the organic solvent under reduced pressure on a rotary evaporator at 30°C in a water bath, and obtained a lipid film on the inner wall of the round-bottom flask. The round-bottom flask containing the lipid film was dried in a vacuum dryer for 18 h to completely remove the residual organic solvent, 4 mL of ultrapure water was added to the lipid film, and ultrasonicated at 300 W for 10 min in a 60°C water bath to obtain a hydrated liposome solution, and the hydrated liposome solution was passed through a 0.22 μm polycarbonate membrane twice using a syringe to obtain a liposome solution;

[0059] (123) 2 mL of a 10 mg / mL dye aqueous solution was mixed with 5 mL of a 1 mg / mL modified GO aqueous solution obtained in step (121), and stirred at 100 rpm for 3 h to obtain a dye-GO solution. The dye-GO solution was mixed with 20 mL of the liposome solution obtained in step (122), and ultrasonically treated at 200 W for 8 h in an ice-water bath at 0 °C. The mixture was centrifuged at 3000 rpm for 15 min, and the supernatant was centrifuged five times through an ultrafiltration tube with a molecular weight cutoff of 100 kDa to obtain liposomes@modified GO-fluorescent dye. The scanning electron microscopy image is shown in FIG. Figure 1 As shown, the dispersion is good and the particle size is uniform;

[0060] (13) Preparation of liposome fluorescently labeled antibodies:

[0061] (131) S-SMCC was dissolved in sterile water to prepare a 10 mg / mL S-SMCC solution. The liposome@modified GO-fluorescent dye obtained in step (123) was dispersed in ultrapure water at an amount of 15 mg / mL to obtain a liposome@modified GO-fluorescent dye dispersion. 5 μL of the S-SMCC solution was added to 50 μL of the dispersion and reacted at 25 °C for 2 h.

[0062] (132) After the reaction in step (131), 55 μL of the solution was transferred to an ultrafiltration centrifuge tube, and then 500 μL of PBS (containing 0.25 mM EDTA) buffer was added. The solution was centrifuged at 12,000 g for 5 min and the filtrate was removed. Then, 500 μL of PBS (containing 0.25 mM EDTA) buffer was added and mixed and centrifuged. This operation was repeated 5 times for desalting. The desalted product was collected and diluted to 50 μL with pure water to obtain a liposome@modified GO-fluorescent dye-S-SMCC solution.

[0063] (133) MPO antibody was diluted to 2 mg / mL with PBS to obtain antibody solution. Sulfo-LC-SPDP was dissolved in PBS to a concentration of 0.05 mg / mL. Sulfo-LC-SPDP solution was added to 120 μL of antibody solution to make the final concentration of Sulfo-LC-SPDP 1.5 μg / mL. PBS was added to the antibody concentration to 0.5 mg / mL. The mixture was reacted at 25 °C for 1 h. The mixture was transferred to an ultrafiltration centrifuge tube and 500 μL of PBS (containing 0.25 mM EDTA) buffer was added. The mixture was centrifuged at 12,000 g for 5 min and the filtrate was removed. 500 μL of PBS (containing 0.25 mM EDTA) buffer was added and mixed and centrifuged. This operation was repeated 5 times. The volume was adjusted to 50 μL with pure water to obtain antibody solution.

[0064] (134) Dissolve TCEP in PBS to a final concentration of 0.05 mg / mL, add the TCEP solution to 50 μL of the antibody solution obtained in step (133) to make a final TCEP concentration of 1 μg / mL, react at 25°C in the dark for 1 h, transfer to an ultrafiltration centrifuge tube, add 500 μL of PBS (containing 0.25 mM EDTA) buffer, centrifuge at 12,000 g for 5 min, remove the filtrate, add 500 μL of PBS (containing 0.25 mM EDTA) buffer, mix and centrifuge, repeat this operation 5 times, and make the volume 50 μL to obtain a McAb-SH intermediate solution;

[0065] (135) 70 μL of the liposome@modified GO-fluorescent dye-S-SMCC solution obtained in step (132) and step (134) and 50 μL of the McAb-SH intermediate solution were mixed evenly and reacted at 25 °C in the dark for 3 h. After the reaction was completed, 0.1 mg / mL PBS (containing 1% BSA, 0.3 mg / mL purslane extract, and 0.2 mg / mL neem extract) solution was added to the reaction system to make the final antibody concentration 0.1 mg / mL to obtain MPO liposome fluorescently labeled antibody solution;

[0066] The preparation steps of the purslane and neem extracts are as follows: wash the purslane and neem, dry them at 80°C, crush them through a 100-mesh sieve, add them into 10 times the mass volume of deionized water, heat them in a water bath at 80°C, stir them at 500 rpm for 2 h, centrifuge them at 8000 rpm for 5 min, filter them, continue the above heating and stirring for the filtrate for 6 h, centrifuge them at 8000 rpm for 5 min, filter them through a 0.22 μm micromembrane, and freeze-dry them at -20°C for 12 h to obtain the extract;

[0067] (14) preparing a mixed fluorescent antibody staining solution: preparing MPO, CitH3 and CD66c liposome fluorescently labeled antibody solutions by the method of step (13), and mixing them evenly in a volume ratio of 0.8:0.6:0.5 to prepare a mixed fluorescent antibody staining solution with a color matching of MPO-FITC, CitH3-PE and CD66c-APC;

[0068] (2) Dissolve BSA, Proclin 300 and tween 20 in PBS buffer solution at mass fractions of 1%, 0.1% and 1% to obtain a washing solution. Centrifuge at 400 g for 5 min after incubation in step (1), discard the supernatant, wash three times with 1000 μL of the washing solution, centrifuge at 400 g for 5 min, and discard the supernatant.

[0069] (3) Add 500 μL of the washing solution obtained in step (2) to resuspend the cells;

[0070] (4) Detection by flow cytometry.

[0071] Example 2: This example provides a method for rapidly detecting neutrophil extracellular traps, comprising the following steps:

[0072] (1) Accurately weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium dihydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate and dissolve them in distilled water. Adjust the pH value to 7.4 with hydrochloric acid and add distilled water to make up to 1000 mL to obtain a hemolytic agent. Take 1000 μL of human whole blood sample, add 1000 μL of the hemolytic agent, and then add 100 μL of the mixed fluorescent antibody staining solution. After vortexing and mixing, incubate at room temperature for 30 min.

[0073] In step (1), the method for preparing the mixed fluorescent antibody staining solution comprises the following steps:

[0074] (11) Preparation of modified eight-arm polyethylene glycol:

[0075] (111) A mixed solution of 0.5 mL of anhydrous ethanol and deionized water (v:v = 2:1) was ultrasonically degassed for 10 min, and 10 mg of 10 kDa eight-arm polyethylene glycol hydroxy and 1 mg of 3-aminopropyltriethoxysilane (APTES) were mixed and added to the ultrasonically degassed mixed solution of anhydrous ethanol and deionized water. The mixture was ultrasonically treated at 200 W for 20 min under N 2 protection, and then ammonia was added to adjust the pH value to 8. The mixture was shaken at 300 rpm and 50 °C for 3 h to obtain a mixed solution.

[0076] (112) Add 2.5 mg of chlorohexane to the mixed solution obtained in step (111), heat to 80°C, stir at 120 rpm for 6 h, stand at 4°C for 4 h, centrifuge at 10,000 rpm for 15 min at 4°C, collect the precipitate, wash repeatedly with acetone, anhydrous ethanol, and deionized water, and dry in a vacuum drying oven at 50°C to constant weight to obtain modified eight-arm polyethylene glycol;

[0077] (12) Preparation of liposomes@modified GO-fluorescent dye:

[0078] (121) Graphene oxide (GO) was prepared by the Hummers method. The graphene oxide solid sample was dispersed in distilled water (1 mg / mL). The GO solution was obtained by intermittent ultrasonication at 600 W for 2 h (on 1 s, off 2 s) using a cell crusher. 80 mL of the treated GO solution was added to 16 mL of 2 mg / mL EDC solution. The solution was ultrasonicated at 570 W for 30 min (on 1.5 s, off 1 s). 300 mg of the modified eight-arm polyethylene glycol obtained in step (112) was added. The solution was ultrasonicated at 300 W for 30 min, stirred at 180 rpm for 12 h, and centrifuged at 8000 rpm for 30 min. The precipitate was washed with deionized water and dried in a vacuum at 25 °C to obtain the modified GO.

[0079] (122) 1,2-Dimyristoylphosphatidylglycerol (DMPG) and dipalmitoylphosphatidylethanolamine (DPPE) were dissolved in 3 mL of chloroform / methanol solution (V 2.5) at a molar ratio of 4:1 (total amount of substance was 3 μmol). 氯仿 :V 甲醇 =2:1), placed in a round-bottom flask, evaporated the organic solvent under reduced pressure on a rotary evaporator at 30°C in a water bath, and obtained a lipid film on the inner wall of the round-bottom flask. The round-bottom flask containing the lipid film was dried in a vacuum dryer for 16 h to completely remove the residual organic solvent, 4 mL of ultrapure water was added to the lipid film, and ultrasonicated at 300 W for 10 min at 60°C in a water bath to obtain a hydrated liposome solution, and the hydrated liposome solution was passed through a 0.22 μm polycarbonate membrane twice using a syringe to obtain a liposome solution;

[0080] (123) 2 mL of a 10 mg / mL dye aqueous solution was mixed with 5 mL of a 1 mg / mL modified GO aqueous solution obtained in step (121), and stirred at 100 rpm for 2 h to obtain a dye-GO solution. The dye-GO solution was mixed with 20 mL of the liposome solution obtained in step (122), and ultrasonically treated at 200 W for 8 h in an ice-water bath at 0 °C, and centrifuged at 3000 rpm for 15 min. The supernatant was centrifuged three times through an ultrafiltration tube with a molecular weight cutoff of 100 kDa to obtain liposomes@modified GO-fluorescent dye.

[0081] (13) Preparation of liposome fluorescently labeled antibodies:

[0082] (131) S-SMCC was dissolved in sterile water to prepare a 10 mg / mL S-SMCC solution. The liposome@modified GO-fluorescent dye obtained in step (123) was dispersed in ultrapure water at an amount of 15 mg / mL to obtain a liposome@modified GO-fluorescent dye dispersion. 5 μL of the S-SMCC solution was added to 50 μL of the dispersion and reacted at 25 °C for 1 h.

[0083] (132) After the reaction in step (131), 55 μL of the solution was transferred to an ultrafiltration centrifuge tube, and then 500 μL of PBS (containing 0.25 mM EDTA) buffer was added. The solution was centrifuged at 12,000 g for 5 min and the filtrate was removed. Then, 500 μL of PBS (containing 0.25 mM EDTA) buffer was added and mixed and centrifuged. This operation was repeated 5 times for desalting. The desalted product was collected and diluted to 50 μL with pure water to obtain a liposome@modified GO-fluorescent dye-S-SMCC solution.

[0084] (133) MPO antibody was diluted to 2 mg / mL with PBS to obtain antibody solution. Sulfo-LC-SPDP was dissolved in PBS to a concentration of 0.05 mg / mL. Sulfo-LC-SPDP solution was added to 120 μL of antibody solution to make the final concentration of Sulfo-LC-SPDP 1.5 μg / mL. PBS was added to the antibody concentration to 0.5 mg / mL. The mixture was reacted at 25 °C for 1 h. The mixture was transferred to an ultrafiltration centrifuge tube and 500 μL of PBS (containing 0.25 mM EDTA) buffer was added. The mixture was centrifuged at 12,000 g for 5 min and the filtrate was removed. 500 μL of PBS (containing 0.25 mM EDTA) buffer was added and mixed and centrifuged. This operation was repeated 5 times. The volume was adjusted to 50 μL with pure water to obtain antibody solution.

[0085] (134) Dissolve TCEP in PBS to a final concentration of 0.05 mg / mL, add the TCEP solution to 50 μL of the antibody solution obtained in step (133) to make a final TCEP concentration of 1 μg / mL, react at 25°C in the dark for 1 h, transfer to an ultrafiltration centrifuge tube, add 500 μL of PBS (containing 0.25 mM EDTA) buffer, centrifuge at 12,000 g for 5 min, remove the filtrate, add 500 μL of PBS (containing 0.25 mM EDTA) buffer, mix and centrifuge, repeat this operation 5 times, and make the volume 50 μL to obtain a McAb-SH intermediate solution;

[0086] (135) 40 μL of the liposome@modified GO-fluorescent dye-S-SMCC solution obtained in step (132) and step (134) and 50 μL of the McAb-SH intermediate solution were mixed evenly and reacted at 25 °C in the dark for 3 h. After the reaction was completed, 0.1 mg / mL PBS (containing 1% BSA, 0.3 mg / mL purslane extract, and 0.2 mg / mL neem extract) solution was added to the reaction system to make the final antibody concentration 0.1 mg / mL to obtain MPO liposome fluorescently labeled antibody solution;

[0087] The preparation steps of purslane and neem extracts are as follows: wash purslane and neem, dry at 80°C, crush through a 100-mesh sieve, add into 10 times the mass volume of deionized water, heat in a water bath at 70°C, stir at 400 rpm for 1 hour, centrifuge at 8000 rpm for 5 minutes and filter, continue heating and stirring the filtrate for 6 hours, centrifuge at 8000 rpm for 5 minutes, filter through a 0.22 μm micromembrane, and freeze-dry at -20°C for 12 hours to obtain the extract;

[0088] (14) preparing a mixed fluorescent antibody staining solution: preparing MPO, CitH3, CD66c and CD45 liposome fluorescently labeled antibody solutions by the method of step (13), and mixing them evenly in a volume ratio of 1.5:1.2:1:1 to prepare a mixed fluorescent antibody staining solution with a color matching of MPO-FITC, CitH3-PE, CD66c-PreCP and CD45-APC;

[0089] (2) Dissolve BSA, Proclin 300 and tween 20 in PBS buffer solution at mass fractions of 1%, 0.1% and 1% to obtain a washing solution. Centrifuge at 400 g for 5 min after incubation in step (1), discard the supernatant, wash three times with 1000 μL of the washing solution, centrifuge at 400 g for 5 min, and discard the supernatant.

[0090] (3) Add 500 μL of the washing solution obtained in step (2) to resuspend the cells;

[0091] (4) Detection by flow cytometry.

[0092] Example 3: This example provides a method for rapidly detecting neutrophil extracellular traps, comprising the following steps:

[0093] (1) Accurately weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium dihydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate and dissolve them in distilled water. Adjust the pH value to 7.4 with hydrochloric acid and add distilled water to make up to 1000 mL to obtain a hemolytic agent. Take 1000 μL of human whole blood sample, add 1000 μL of the hemolytic agent, and then add 100 μL of the mixed fluorescent antibody staining solution. After vortexing and mixing, incubate at room temperature for 30 min.

[0094] In step (1), the method for preparing the mixed fluorescent antibody staining solution comprises the following steps:

[0095] (11) Preparation of modified eight-arm polyethylene glycol hydroxyl group:

[0096] (111) A mixed solution of 0.5 mL of anhydrous ethanol and deionized water (v:v = 2:1) was ultrasonically degassed for 10 min, and 10 mg of 10 kDa eight-arm polyethylene glycol hydroxy and 1 mg of 3-aminopropyltriethoxysilane (APTES) were mixed and added to the ultrasonically degassed mixed solution of anhydrous ethanol and deionized water. The mixture was ultrasonically treated at 200 W for 20 min under N 2 protection, and then ammonia was added to adjust the pH value to 8.5. The mixture was shaken at 300 rpm and 50 °C for 3 h to obtain a mixed solution.

[0097] (112) Chlorohexane was added to the mixed solution obtained in step (111), the temperature was raised to 80°C, and the mixture was stirred at 120 rpm for 7 h. The mixture was allowed to stand at 4°C for 5 h, and centrifuged at 10,000 rpm for 15 min at 4°C. The precipitate was collected, and washed repeatedly with acetone, anhydrous ethanol, and deionized water. The precipitate was dried in a vacuum oven at 50°C to constant weight to obtain modified eight-arm polyethylene glycol.

[0098] (12) Preparation of liposomes@modified GO-fluorescent dye:

[0099] (121) Graphene oxide (GO) was prepared by the Hummers method. The graphene oxide solid sample was dispersed in distilled water (1 mg / mL). The GO solution was obtained by intermittent ultrasonication at 600 W for 2 h (on 1 s, off 2 s) using a cell crusher. 80 mL of the treated GO solution was added to 16 mL of 2 mg / mL EDC solution. The solution was ultrasonicated at 570 W for 30 min (on 1.5 s, off 1 s). 300 mg of the modified eight-arm polyethylene glycol obtained in step (112) was added. The solution was ultrasonicated at 300 W for 30 min, stirred at 180 rpm for 12 h, and centrifuged at 8000 rpm for 30 min. The precipitate was washed with deionized water and dried in a vacuum at 25 °C to obtain the modified GO.

[0100] (122) 1,2-Dimyristoylphosphatidylglycerol (DMPG) and dipalmitoylphosphatidylethanolamine (DPPE) were dissolved in 3 mL of chloroform / methanol solution (V 2.5) at a molar ratio of 4:1 (total amount of substance was 3 μmol). 氯仿 :V 甲醇 =2:1), placed in a round-bottom flask, and the organic solvent was evaporated under reduced pressure on a rotary evaporator at 30°C in a water bath to obtain a lipid film on the inner wall of the round-bottom flask. The round-bottom flask containing the lipid film was dried in a vacuum dryer for 17 h to completely remove the residual organic solvent, 4 mL of ultrapure water was added to the lipid film, and ultrasonicated at 300 W for 10 min in a 60°C water bath to obtain a hydrated liposome solution, and the hydrated liposome solution was passed through a 0.22 μm polycarbonate membrane twice using a syringe to obtain a liposome solution;

[0101] (123) 2 mL of a 10 mg / mL dye aqueous solution was mixed with 5 mL of a 1 mg / mL modified GO aqueous solution obtained in step (121), and stirred at 100 rpm for 2.5 h to obtain a dye-GO solution. The dye-GO solution was mixed with 20 mL of the liposome solution obtained in step (122), and ultrasonically treated at 200 W for 8 h in an ice-water bath at 0 °C. The mixture was centrifuged at 3000 rpm for 15 min, and the supernatant was centrifuged four times through an ultrafiltration tube with a molecular weight cutoff of 100 kDa to obtain liposomes@modified GO-fluorescent dye.

[0102] (13) Preparation of liposome fluorescently labeled antibodies:

[0103] (131) S-SMCC was dissolved in sterile water to prepare a 10 mg / mL S-SMCC solution. The liposome@modified GO-fluorescent dye obtained in step (123) was dispersed in ultrapure water at an amount of 15 mg / mL to obtain a liposome@modified GO-fluorescent dye dispersion. 5 μL of the S-SMCC solution was added to 50 μL of the dispersion and reacted at 25 °C for 1.5 h.

[0104] (132) After the reaction in step (131), 55 μL of the solution was transferred to an ultrafiltration centrifuge tube, and then 500 μL of PBS (containing 0.25 mM EDTA) buffer was added. The solution was centrifuged at 12,000 g for 5 min and the filtrate was removed. Then, 500 μL of PBS (containing 0.25 mM EDTA) buffer was added and mixed and centrifuged. This operation was repeated 5 times for desalting. The desalted product was collected and diluted to 50 μL with pure water to obtain a liposome@modified GO-fluorescent dye-S-SMCC solution.

[0105] (133) MPO antibody was diluted to 2 mg / mL with PBS to obtain antibody solution. Sulfo-LC-SPDP was dissolved in PBS to a concentration of 0.05 mg / mL. Sulfo-LC-SPDP solution was added to 120 μL of antibody solution to make the final concentration of Sulfo-LC-SPDP 1.5 μg / mL. PBS was added to the antibody concentration to 0.5 mg / mL. The mixture was reacted at 25 °C for 1 h. The mixture was transferred to an ultrafiltration centrifuge tube and 500 μL of PBS (containing 0.25 mM EDTA) buffer was added. The mixture was centrifuged at 12,000 g for 5 min and the filtrate was removed. 500 μL of PBS (containing 0.25 mM EDTA) buffer was added and mixed and centrifuged. This operation was repeated 5 times. The volume was adjusted to 50 μL with pure water to obtain antibody solution.

[0106] (134) Dissolve TCEP in PBS to a final concentration of 0.05 mg / mL, add the TCEP solution to 50 μL of the antibody solution obtained in step (133) to make a final TCEP concentration of 1 μg / mL, react at 25°C in the dark for 1 h, transfer to an ultrafiltration centrifuge tube, add 500 μL of PBS (containing 0.25 mM EDTA) buffer, centrifuge at 12,000 g for 5 min, remove the filtrate, add 500 μL of PBS (containing 0.25 mM EDTA) buffer, mix and centrifuge, repeat this operation 5 times, and make the volume 50 μL to obtain a McAb-SH intermediate solution;

[0107] (135) 50 μL of the liposome@modified GO-fluorescent dye-S-SMCC solution obtained in step (132) and step (134) and 50 μL of the McAb-SH intermediate solution were mixed evenly and reacted at 25 °C in the dark for 3 h. After the reaction was completed, 0.1 mg / mL PBS (containing 1% BSA, 0.3 mg / mL purslane extract, and 0.2 mg / mL neem extract) solution was added to the reaction system to make the final antibody concentration 0.1 mg / mL to obtain MPO liposome fluorescently labeled antibody solution;

[0108] The preparation steps of the purslane and neem extracts are as follows: wash the purslane and neem, dry them at 80°C, crush them through a 100-mesh sieve, add them into 10 times the mass volume of deionized water, heat them in a water bath at 75°C, stir them at 450 rpm for 1.5 h, centrifuge them at 8000 rpm for 5 min, filter them, continue the above heating and stirring for the filtrate again for 6 h, centrifuge them at 8000 rpm for 5 min, filter them through a 0.22 μm micromembrane, and freeze-dry them at -20°C for 12 h to obtain the extract;

[0109] (14) preparing a mixed fluorescent antibody staining solution: preparing MPO, CitH3, CD66c and CD45 liposome fluorescently labeled antibody solutions by the method of step (13), and mixing them evenly in a volume ratio of 1.2:1:0.8:0.2 to prepare a mixed fluorescent antibody staining solution with a color matching of MPO-FITC, CitH3-APC, CD66c-PE and CD45-PreCP;

[0110] (2) Dissolve BSA, Proclin 300 and tween 20 in PBS buffer solution at mass fractions of 1%, 0.1% and 1% to obtain a washing solution. Centrifuge at 400 g for 5 min after incubation in step (1), discard the supernatant, wash three times with 1000 μL of the washing solution, centrifuge at 400 g for 5 min, and discard the supernatant.

[0111] (3) Add 500 μL of the washing solution obtained in step (2) to resuspend the cells;

[0112] (4) Detection by flow cytometry.

[0113] The only difference between Comparative Example 1 and Example 1 is that the modified eight-arm polyethylene glycol is replaced by the eight-arm polyethylene glycol hydroxyl group.

[0114] The only difference between Comparative Example 2 and Example 1 is that the antibody and the fluorescent dye are directly coupled using conventional methods in the art.

[0115] The only difference between Comparative Example 3 and Example 1 is that the purslane and neem extracts are not added.

[0116] Experimental Example 1:

[0117] Sample source: Shanghai Oriental Hospital;

[0118] Sample information: 10 samples from inflammatory patients and 10 samples from normal subjects;

[0119] Detection method: The method of Example 1 of the present invention was used for testing. The color matching of the kit was MPO-FITC, CitH3-PE, CD66c-APC. CD66c positivity was used as the gating condition to test the positive rates of MPO and CitH3. The results are shown in Table 1. Representative flow cytometry staining images are shown in Table 1. Figure 2 shown.

[0120] Table 1: Positive rate of MPO and CitH3 (%)

[0121]

[0122] The results in Table 1 show that the rates of MPO single positivity, CitH3 single positivity, and MPO and CitH3 double positivity in inflammatory patients are significantly higher than those in normal people, which is consistent with the clinical test results, indicating that the method for rapid detection of neutrophil extracellular traps of the present invention is effective and can be used to detect neutrophil extracellular traps and can be applied to auxiliary diagnosis of diseases.

[0123] Experimental Example 2: The same pneumonia patient sample was taken and the methods of Examples 1-3 and Comparative Examples 1-3 were used to perform 10 repeated tests, respectively, to perform repeatability tests, and the coefficient of variation CV (%) was calculated. The results are shown in Figure 3 shown.

[0124] Figure 3 The results show that the method of the present invention was used to perform 10 repeated tests on the sample. The detection repeatability CV of the method of Examples 1-3 was less than 10%, and the repeatability of the method of Comparative Examples 1-3 was worse than that of Examples 1-3, indicating that the test paper of the present invention has high detection accuracy and good repeatability. In addition, the mixed fluorescent antibody staining solution prepared in Examples 1-3 and Comparative Examples 1-3 was stored at 4°C for 12 months. The CV results are as follows: Figure 4 As shown, there is no significant change in Examples 1-3 compared with before storage, and the change in Comparative Examples 1-3 is significantly greater than that in Examples 1-3, indicating that the detection method of the present invention has good stability.

[0125] Experimental Example 3: 1 mg of liposomes@modified GO-fluorescent dye (FITC) prepared in Examples 1-3 and Comparative Example 1 was dispersed in 1 mL of ultrapure water, and the fluorescence intensity at the maximum emission wavelength was detected. At the same time, 1 mg of FITC was dispersed in 1 mL of ultrapure water as a control group, and irradiated with a 100W xenon lamp as the excitation light source. The distance between the sample and the xenon lamp was 5 cm. After 60 minutes, the fluorescence intensity at the maximum emission wavelength was detected again, and the fluorescence intensity decrease rate before and after light irradiation was calculated. Take 1 mg of liposomes@modified GO-fluorescent dye (FITC) of Examples 1-3 and Comparative Example 1 and disperse it in 1 mL of ultrapure water, and use a fluorescence spectrophotometer to detect the fluorescence intensity at an excitation wavelength of 492 nm. Ultrasonicate for 12 h, centrifuge at 10000 rpm for 30 min, discard the supernatant, resuspend with 1 mL of water, and use an F-2500 fluorescence spectrophotometer to detect the fluorescence intensity at an excitation wavelength of 492 nm after dispersion. The fluorescence intensity decrease rate (%) before and after ultrasound is calculated, and the results are shown in Table 2. The liposome fluorescent (FITC) labeled antibody solution or the fluorescent labeled antibody solution directly coupled with the fluorescent dye prepared in Examples 1-3 and Comparative Examples 1-3 were taken to detect the fluorescence intensity. The fluorescence intensity was detected again after being stored at 4°C for 12 months, and the fluorescence intensity decrease rate was calculated. The results are as follows: Figure 5 shown.

[0126] Table 2: Fluorescence intensity decrease rate

[0127]

[0128] The results in Table 2 show that after 60 min of xenon lamp irradiation, the fluorescence intensity of Examples 1-3 decreased by less than 3%, which was significantly lower than that of Comparative Example 1, indicating that the liposome@modified GO-fluorescent dye has good photostability, can protect the dye, effectively reduce the photobleaching effect of external light sources on the fluorescent molecules, and significantly improve its anti-light stability. At the same time, the liposome@modified GO-fluorescent dye is not prone to dye leakage in the solution and has strong stability. Figure 5 The results showed that the fluorescence intensity decrease rate of Comparative Examples 1-3 after storage was significantly higher than that of Examples 1-3, indicating that the liposome fluorescent (FITC) labeled antibody of the present invention has strong storage stability.

[0129] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

Claims

1. A method for rapid detection of neutrophil extracellular traps, characterized in that: The following steps are involved: (1) Take a whole blood sample, add a hemolytic agent, then add a mixed fluorescent antibody staining solution, shake and mix, and incubate at room temperature; (2) After incubation in step (1), centrifuge and discard the supernatant; wash with a washing solution and then centrifuge and discard the supernatant; (3) Add washing buffer to resuspend the cells; (4) Detection by flow cytometry; In step (1), the method for preparing the mixed fluorescent antibody staining solution comprises the following steps: (11) Preparation of modified eight-arm polyethylene glycol: (111) ultrasonically degassing a mixed solution of anhydrous ethanol and deionized water, mixing eight-arm polyethylene glycol hydroxyl and APTES, adding the mixture to the ultrasonically degassed mixed solution of anhydrous ethanol and deionized water, ultrasonically treating under N2 protection, adding ammonia water to adjust the pH value, and shaking to obtain a mixed solution; (112) adding chlorohexane to the mixed solution obtained in step (111), heating, stirring, standing, centrifuging, washing the precipitate, and drying to obtain a modified eight-arm polyethylene glycol; (12) Preparation of liposomes@modified GO-fluorescent dye: (121) dispersing the graphene oxide solid sample into distilled water, intermittently sonicating to obtain a treated GO solution, taking the treated GO solution and adding it to the solution, sonicating, taking the modified eight-arm polyethylene glycol obtained in step (112) and adding it, sonicating, stirring, centrifuging, washing the precipitate, and drying to obtain modified GO; (122) DMPG and DPPE were dissolved in chloroform / methanol solution, and the organic solvent was evaporated under reduced pressure by rotary evaporation. The solution was dried, ultrapure water was added, and water bath sonication was performed to obtain a hydrated liposome solution, which was then passed through a 0.22 μm membrane to obtain a liposome solution; (123) mixing a 10 mg / mL dye aqueous solution with a 1 mg / mL modified GO aqueous solution obtained in step (121), stirring to obtain a dye-GO solution, mixing the dye-GO solution with the liposome solution obtained in step (122), ultrasonically treating in an ice water bath, centrifuging, and ultrafiltrating the supernatant to obtain liposomes@modified GO-fluorescent dye; (13) Preparation of liposome fluorescently labeled antibodies: (131) dissolving S-SMCC in sterile water to prepare a 10 mg / mL S-SMCC solution, dispersing the liposome@modified GO-fluorescent dye obtained in step (123) in ultrapure water at an amount of 15 mg / mL to obtain a liposome@modified GO-fluorescent dye dispersion, and adding the S-SMCC solution to the dispersion for reaction; (132) After the reaction in step (131) is completed, PBS buffer containing EDTA is added for desalting, the desalted product is collected, and the volume is fixed with pure water to obtain a liposome@modified GO-fluorescent dye-S-SMCC solution; (133) The MPO antibody was diluted to 2 mg / mL with PBS to obtain an antibody solution, Sulfo-LC-SPDP was dissolved with PBS to a concentration of 0.05 mg / mL, Sulfo-LC-SPDP solution was added to the antibody solution to make the final concentration of Sulfo-LC-SPDP 1.5 μg / mL, PBS was added to the antibody concentration to 0.5 mg / mL, the solution was desalted, and the antibody solution was fixed to volume with pure water to obtain an antibody fixed solution; (134) Dissolve TCEP in PBS to a final concentration of 0.05 mg / mL, add the TCEP solution to the antibody solution obtained in step (133) to make the final TCEP concentration 1 μg / mL, protect from light, desalt, and adjust the volume to obtain a McAb-SH intermediate solution; (135) The liposome@modified GO-fluorescent dye-S-SMCC solution and the McAb-SH intermediate solution obtained in step (132) and step (134) are mixed evenly and reacted in the dark. After the reaction is completed, a 0.1 mg / mL PBS solution containing 1% BSA, 0.3 mg / mL purslane extract, and 0.2 mg / mL neem extract is added to the reaction system to make the final antibody concentration 0.1 mg / mL to obtain an MPO liposome fluorescently labeled antibody solution; (14) preparing a mixed fluorescent antibody staining solution: preparing MPO, CitH3, CD66c and CD45 liposome fluorescently labeled antibody solutions by the method of step (13), and mixing them evenly to form a mixed fluorescent antibody staining solution; In step (135), the preparation steps of the purslane and neem extract are as follows: wash the purslane and neem, dry them at 80°C, crush them through a 100-mesh sieve, add them into 10 times the mass volume of deionized water, heat them in a water bath at 70-80°C, stir them at 400-500 rpm for 1-2 h, centrifuge them at 8000 rpm for 5 min, filter them, continue the above heating and stirring for the filtrate for 6 h, centrifuge them at 8000 rpm for 5 min, filter them through a 0.22 μm micromembrane, and freeze-dry them at -20°C for 12 h to obtain the extract.

2. The method for rapid detection of neutrophil extracellular traps according to claim 1, characterized in that: In step (111), the ratio of anhydrous ethanol, eight-arm polyethylene glycol hydroxyl group and APTES is 0.5 mL:10 mg:1 mg.

3. The method for rapid detection of neutrophil extracellular traps according to claim 2, characterized in that In step (112), the mass ratio of the chlorohexane to the eight-arm polyethylene glycol hydroxyl group in step (111) is 1:

4.

4. The method for rapid detection of neutrophil extracellular traps according to claim 3, characterized in that: In step (121), the volume ratio of the treated GO solution to the 2 mg / mL EDC solution is 5:1; the dosage ratio of the modified eight-arm polyethylene glycol to the treated GO solution is 15 mg:4 mL.

5. The method for rapid detection of neutrophil extracellular traps according to claim 4, characterized in that: In step (122), the molar ratio of DMPG to DPPE is 4:1; the dosage ratio of DMPG to chloroform / methanol solution is 0.8 μmol:1 mL; and the volume ratio of ultrapure water to chloroform / methanol solution is 4:

3.

6. The method for rapid detection of neutrophil extracellular traps according to claim 5, characterized in that: In step (123), the volume ratio of the dye aqueous solution, the modified GO aqueous solution and the liposome solution is 2:5:

20.

7. The method for rapid detection of neutrophil extracellular traps according to claim 6, characterized in that: In step (131), the volume ratio of the liposome@modified GO-fluorescent dye dispersion to the S-SMCC solution is 10:

1.

8. The method for rapid detection of neutrophil extracellular traps according to claim 7, characterized in that: In step (135), the volume ratio of the liposome@modified GO-fluorescent dye-S-SMCC solution to the McAb-SH intermediate solution is 4-7:

5.

9. The method for rapid detection of neutrophil extracellular traps according to claim 8, characterized in that: In step (14), the volume ratio of the MPO, CitH3, CD66c and CD45 liposome fluorescently labeled antibody solution is 0.8-1.5:0.6-1.2:0.5-1:0-1.

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