A multimodal nanoprobes NETs detection kit, a preparation method thereof and a method for detecting concentration of NETs

Abstract

The application belongs to the field of biomedical detection, and relates to a multi-modal nano probe NETs detection kit, a preparation method thereof and a NETs concentration detection method. The multi-modal nano probe NETs detection kit comprises a nano probe and a chemiluminescence working solution. The nano probe comprises a gold nano star, a histone H3 antibody-glucose oxidase conjugate fixed on the surface of the gold nano star, an MPO antibody-horseradish peroxidase conjugate and a double-stranded DNA aptamer-alkaline phosphatase conjugate. The chemiluminescence working solution is a Tris-HCl buffer solution containing glucose, luminol, p-nitrophenyl phosphate and an enhancer. In the application, after the nano probe is combined with NETs, the addition of the chemiluminescence working solution can form an efficient signal amplification network, the detection sensitivity of the kit to NETs can reach 0.02 nM, and low-concentration NETs of early inflammatory patients can be accurately detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection, and in particular to a multi-modal nanoprobe NETs detection kit, a preparation method thereof and a NETs concentration detection method. BACKGROUND

[0002] Neutrophil extracellular traps (NETs) are DNA-protein complex structures released by activated neutrophils, mainly composed of chromatin DNA skeleton, histone (such as H3), myeloperoxidase (MPO), elastase, etc., and play an important role in host defense, inflammatory response and various diseases such as sepsis, rheumatoid arthritis, thrombosis, tumor metastasis, etc. Abnormal elevation of NETs level has become a biomarker of various inflammatory diseases, and its accurate detection has important clinical value for early diagnosis, efficacy evaluation and prognosis of diseases.

[0003] At present, the commonly used NETs detection kits in clinical practice mainly include ELISA kits, immunofluorescence staining kits, etc. However, these kits and detection methods generally have problems such as low sensitivity, complex operation, strong equipment dependence, etc.

[0004] ELISA kits can usually only detect single components such as MPO-DNA complexes, and have a high lower limit of detection, making it difficult to meet the detection needs of low-concentration samples. Although immunofluorescence staining kits can achieve multi-target labeling, they have problems such as spontaneous fluorescence interference, serious photobleaching, and the need for expensive fluorescence microscopes.

[0005] Therefore, there is an urgent need to develop a new NETs detection kit with high sensitivity. SUMMARY

[0006] (I) Technical problems to be solved

[0007] In order to solve the problem of low sensitivity of the existing NETs detection kit, the present application provides a multi-modal nanoprobe NETs detection kit, a preparation method thereof and a NETs concentration detection method.

[0008] (II) Technical solutions

[0009] In order to achieve the above purpose, the main technical solutions adopted by the present application include:

[0010] A multi-modal nano-probe NETs detection kit, comprising a nano-probe and a chemiluminescence working solution, wherein the nano-probe comprises a gold nano-star, a histone H3 antibody-glucose oxidase conjugate, a MPO antibody-horseradish peroxidase conjugate and a double-stranded DNA aptamer-alkaline phosphatase conjugate fixed on the surface of the gold nano-star;

[0011] The histone H3 antibody-glucose oxidase conjugate is a complex of an anti-histone H3 antibody and glucose oxidase formed by chemical cross-linking, the MPO antibody-horseradish peroxidase conjugate is a complex of a MPO antibody and horseradish peroxidase formed by chemical cross-linking, and the double-stranded DNA aptamer-alkaline phosphatase conjugate is a complex of a double-stranded DNA aptamer and alkaline phosphatase formed by chemical cross-linking.

[0012] The chemiluminescence working solution is a Tris-HCl buffer solution containing glucose, luminol, p-nitrophenyl phosphate and an enhancer.

[0013] The multi-modal nano-probe NETs detection kit as described above, preferably, in the chemiluminescence working solution, the enhancer is p-iodophenol, the mass concentration of the p-iodophenol is 0.1-0.2%, the concentration of the glucose is 5-10 mM, the concentration of the luminol is 0.1-0.3 mM, the concentration of the p-nitrophenyl phosphate is 1.0-2.0 mM, and the pH of the Tris-HCl buffer solution is 8.5-9.0.

[0014] The multi-modal nano-probe NETs detection kit as described above, preferably, the particle size of the gold nano-star is 70-90 nm.

[0015] In the histone H3 antibody-glucose oxidase conjugate, the molar ratio of the histone H3 antibody to the glucose oxidase is 1:3-1:5.

[0016] In the MPO antibody-horseradish peroxidase conjugate, the molar ratio of the MPO antibody to the horseradish peroxidase is 1:3-1:5.

[0017] In the double-stranded DNA aptamer-alkaline phosphatase conjugate, the molar ratio of the double-stranded DNA aptamer to the alkaline phosphatase is 1:3-1:5.

[0018] In a second aspect, the present application provides a preparation method of the multi-modal nano-probe NETs detection kit, comprising the following steps:

[0019] S1: dispersing the gold nano-star in a mercaptoundecanoic acid solution, adding mercapto-polyethylene glycol after oscillation treatment, and obtaining a first mixed solution after reaction;

[0020] S2: adding PBS buffer solution to the first mixed solution, and then sequentially adding the histone H3 antibody-glucose oxidase conjugate, the MPO antibody-horseradish peroxidase conjugate, and the double-stranded DNA aptamer-alkaline phosphatase conjugate, and performing incubation treatment;

[0021] S3: after the incubation treatment is completed, adding 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt to the system, and obtaining the nano probe after reaction.

[0022] Preferably, in the preparation method of the multi-modal nano probe NETs detection kit as described above, the concentration of the mercaptoundecanoic acid solution is 0.1-0.2 mM, the mercapto polyethylene glycol is reacted after being added at 4 ℃ for 12-16 h, and the molecular weight of the mercapto polyethylene glycol is 10-12 kDa.

[0023] Preferably, in the preparation method of the multi-modal nano probe NETs detection kit as described above, the molar ratio of the histone H3 antibody, the MPO antibody, and the double-stranded DNA aptamer is 1:1:1 in step S2, and the 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt is reacted at 4 ℃ for 30-40 min after being added in step S3.

[0024] In a third aspect, the present application further provides a method for detecting the concentration of NETs by using the multi-modal nano probe NETs detection kit or the multi-modal nano probe NETs detection kit prepared by the preparation method, which is a method for non-disease diagnosis purposes, and comprises the following steps:

[0025] A1: adding a sample to be detected into the nano probe, and obtaining a probe-NETs complex after incubation treatment;

[0026] A2: adding an equal volume of a polyethylene glycol solution into the probe-NETs complex, uniformly mixing, and then performing centrifugal treatment, and obtaining a precipitate after the supernatant is discarded;

[0027] A3: adding a chemiluminescence working solution into the precipitate, detecting the relative luminescence intensity of the system after dark reaction, and calculating the concentration of NETs.

[0028] Preferably, in the method for detecting the concentration of NETs as described above, the temperature of incubation is 37 ℃, and the incubation time is 15-20 min in step A1.

[0029] Preferably, in the method for detecting the concentration of NETs as described above, the concentration of the polyethylene glycol solution is 8-10 wt% in step A2.

[0030] The method for detecting the concentration of NETs as described above, preferably, in step A3, the volume of the chemiluminescence working solution is the same as that of the probe-NETs complex, the reaction temperature is 37 DEG C, and the reaction time is 5-8 min.

[0031] (III) Beneficial Effects

[0032] Firstly, the present application constructs a multi-target point synergistic recognition system by simultaneously arranging the histone H3 antibody-glucose oxidase conjugate, the MPO antibody-horseradish peroxidase conjugate and the double-stranded DNA aptamer-alkaline phosphatase conjugate on the surface of the gold nanostar, so that the nano probe can capture the histone H3, MPO and dsDNA in the NETs at the same time, and the accuracy of detection can be improved.

[0033] Secondly, in the present application, after the nano probe is combined with the NETs, the addition of the chemiluminescence working solution can form an efficient signal amplification network, the H2O2 generated by the catalysis of glucose oxidase can be immediately utilized by the horseradish peroxidase to catalyze the generation of the initial chemiluminescence signal of luminol, and at the same time, the alkaline phosphatase hydrolyzes the p-nitrophenyl phosphate to generate the p-nitrophenol which cooperates with the p-iodophenol to significantly improve the light intensity. The cascade amplification mechanism of the present application can make the detection sensitivity of the kit for the NETs reach 0.02 nM, and the low concentration of NETs in the early inflammatory patients can be accurately detected. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is a standard curve diagram of the relative light intensity and the concentration of NETs in the present application. DETAILED DESCRIPTION

[0035] In order to better explain the present application, the present application is described in detail below in combination with specific embodiments.

[0036] The present application provides a multi-modal nano probe NETs detection kit, which comprises a nano probe and a chemiluminescence working solution. In the present application, the nano probe and the chemiluminescence working solution are separate parts. The nano probe comprises a gold nanostar, a histone H3 antibody-glucose oxidase conjugate, a MPO antibody-horseradish peroxidase conjugate and a double-stranded DNA aptamer-alkaline phosphatase conjugate which are fixed on the surface of the gold nanostar.

[0037] The histone H3 antibody-glucose oxidase conjugate is a complex formed by the chemical cross-linking of the anti-histone H3 antibody and the glucose oxidase, the MPO antibody-horseradish peroxidase conjugate is a complex formed by the chemical cross-linking of the MPO antibody and the horseradish peroxidase, and the double-stranded DNA aptamer-alkaline phosphatase conjugate is a complex formed by the chemical cross-linking of the double-stranded DNA aptamer and the alkaline phosphatase.

[0038] The chemiluminescence working solution is a Tris-HCl buffer solution containing glucose, luminol, p-nitrophenyl phosphate, and an enhancer.

[0039] The present application constructs a multi-target synergistic recognition system by simultaneously arranging the histone H3 antibody-glucose oxidase conjugate, the MPO antibody-horseradish peroxidase conjugate, and the double-stranded DNA aptamer-alkaline phosphatase conjugate on the surface of the gold nanostar, so that the nanoprobes can simultaneously capture the histone H3 in the NETs, the MPO in the NETs, and the dsDNA (double-stranded DNA) in the NETs, and the accuracy of detection can be improved.

[0040] In the present application, after the nanoprobes are combined with the NETs, the addition of the chemiluminescence working solution can form an efficient signal amplification network. Specifically, the chemiluminescence working solution contains glucose, luminol, p-nitrophenyl phosphate, and an enhancer, wherein the enhancer can be synergistically amplified with the p-nitrophenol generated by the catalysis of the alkaline phosphatase under alkaline conditions. After the fluorescent probes are combined with the NETs, the glucose oxidase catalyzes the glucose to generate H2O2, and the H2O2 oxidizes the luminol under the action of the horseradish peroxidase, so that the luminol generates a light signal. At the same time, the alkaline phosphatase hydrolyzes the p-nitrophenyl phosphate to release the p-nitrophenol, further enhancing the light intensity. The cascade amplification mechanism of the present application can make the detection sensitivity of the kit for the NETs reach 0.02 nM, and can more accurately detect the low concentration of NETs in early inflammatory patients.

[0041] Preferably, in the chemiluminescence working solution, the enhancer is p-iodophenol, the mass concentration of the p-iodophenol is 0.1-0.2%, the concentration of the glucose is 5-10 mM, the concentration of the luminol is 0.1-0.3 mM, the concentration of the p-nitrophenyl phosphate is 1.0-2.0 mM, and the pH of the Tris-HCl buffer solution is 8.5-9.0.

[0042] Preferably, the particle size of the gold nanostar is 70-90 nm. The gold nanostar has a unique star-shaped structure and a high specific surface area, and the surface tips and branches thereof can effectively enhance the local electromagnetic field, which is conducive to improving the enzyme catalytic efficiency. At the same time, the gold nanostar has good biocompatibility and stable gold-sulfur bond combination ability, which helps to maintain the activity of the fixed enzymes and antibodies, and ensures the efficiency and repeatability of signal generation in the detection process.

[0043] The histone H3 antibody-glucose oxidase conjugate is used to recognize histone H3 in the NETs and catalyze glucose to generate H2O2. The MPO antibody-horseradish peroxidase conjugate is used to recognize MPO in the NETs and catalyze luminol to generate chemiluminescence, and the double-stranded DNA aptamer-alkaline phosphatase conjugate is used to recognize dsDNA in the NETs and hydrolyze pNPP (nitrophenyl phosphate) to generate p-nitrophenol, further enhancing the light intensity. In the histone H3 antibody-glucose oxidase conjugate, the molar ratio of the histone H3 antibody to the glucose oxidase is 1:3-1:5, in the MPO antibody-horseradish peroxidase conjugate, the molar ratio of the MPO antibody to the horseradish peroxidase is 1:3-1:5, and in the double-stranded DNA aptamer-alkaline phosphatase conjugate, the molar ratio of the double-stranded DNA aptamer to the alkaline phosphatase is 1:3-1:5.

[0044] The application also provides a preparation method of the above-mentioned multi-modal nano probe NETs detection kit, comprising the following steps:

[0045] S1: disperse the gold nanostar in the mercapto undecanoic acid solution, add mercapto polyethylene glycol after oscillation treatment, and obtain a first mixed solution after reaction.

[0046] S2: add PBS buffer to the first mixed solution, and then sequentially add the histone H3 antibody-glucose oxidase conjugate, the MPO antibody-horseradish peroxidase conjugate, and the double-stranded DNA aptamer-alkaline phosphatase conjugate, and perform incubation treatment.

[0047] S3: after the incubation treatment is completed, add 4-(N-maleimido methyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt to the system, remove the free components therein after reaction, add PBS buffer to make the final concentration of the gold nanostar 4-5 nM, and obtain the nano probe.

[0048] In step S1, the mercapto undecanoic acid (11-mercapto undecanoic acid, 11-Mercaptoundecanoic acid, MUA) has the following functions: forming a stable Au-S bond with the gold atoms (Au) on the surface of the gold nanostar through the terminal mercapto group (-SH) to realize molecular anchoring, and exposing the carboxyl group (-COOH) in the solution to provide a chemical reaction site for subsequent introduction of active groups.

[0049] The molecular weight of the mercapto polyethylene glycol (ω-mercapto-polyethylene glycol) is 8000-12000 Da, and it has the following functions: the PEG chain has strong hydrophilicity and space shielding effect, which can significantly improve the dispersibility and non-specific adsorption capacity of the gold nanostar in the biological sample, prevent the probe from aggregating or non-specifically combining with serum proteins in the complex sample, and improve the detection specificity.

[0050] Preferably, in the step S1, the concentration of the mercaptoundecanoic acid solution is 0.1-0.2 mM, the mercaptopolyethylene glycol is added and reacted at 4℃ for 12-16 h, and the molecular weight of the mercaptopolyethylene glycol is 10-12 kDa.

[0051] Preferably, in the step S2, the molar ratio of the histone H3 antibody, the MPO antibody and the double-stranded DNA aptamer is 1:1:1, and in the step S3, the Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) is added and reacted at 4℃ for 30-40 min.

[0052] In the step S2, the three kinds of conjugates are added in sequence, which can avoid the spatial competition or aggregation caused by the simultaneous addition of multiple macromolecules, and controllably realize the ordered fixation of multiple components and improve the loading uniformity. The purpose of the incubation treatment is to combine the conjugates with the gold nanostar.

[0053] In the step S3, the Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) is a bifunctional crosslinking agent, one end of which is an NHS ester, which can react with the carboxyl of the PEG or MUA on the surface of the gold nanostar to form a stable amide bond, and the other end is a maleimide group, which can undergo Michael addition with the antibody / aptamer in the conjugate to form a stable thioether bond, thereby realizing the covalent connection between the gold nanostar and the three kinds of conjugates, and greatly improving the stability of the nanoprobes.

[0054] The application also provides a method for detecting the concentration of NETs by using the above-mentioned multi-modal nanoprobes NETs detection kit or the multi-modal nanoprobes NETs detection kit prepared by using the above-mentioned preparation method, which comprises the following steps:

[0055] A1: adding a sample to be tested into the nanoprobes, and obtaining a probe-NETs complex after incubation treatment.

[0056] A2: adding an equal volume of a polyethylene glycol solution into the probe-NETs complex, mixing, and then performing centrifugal treatment to obtain a precipitate after discarding the supernatant.

[0057] A3: adding a chemiluminescence working solution into the precipitate, detecting the relative luminescence intensity of the system after dark reaction, and calculating the concentration of NETs.

[0058] Preferably, in the step A1, the temperature of the incubation is 37℃, and the incubation time is 15-20 min, which is enough to complete the cooperative recognition of multiple targets, and can avoid enzyme inactivation or non-specific adsorption, and the sample to be tested is a pretreated serum, plasma, synovial fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, neutrophil culture supernatant or inflammatory tissue homogenate.

[0059] Preferably, in the above-mentioned step A2, the concentration of the polyethylene glycol solution is 8-10 wt%. PEG is a "rejection agent" that can dehydrate, aggregate and precipitate the probe-NETs, while small molecular impurities, such as free enzymes, unbound probes, serum proteins, etc., remain in the solution. Centrifugal treatment can achieve solid-liquid separation, enrich the target complex, significantly reduce the background signal, and improve the signal-to-noise ratio.

[0060] Preferably, in the above-mentioned step A3, the volume of the chemiluminescence working solution is the same as that of the probe-NETs complex, the reaction temperature is 37°C, and the reaction time is 5-8 min.

[0061] In addition, the preparation method of the histone H3 antibody-glucose oxidase conjugate in the present application is as follows:

[0062] The anti-histone H3 antibody is dissolved in PBS buffer, and a sulfo-maleimide methylcyclohexane carboxylic acid succinimidyl ester solution is added to the anti-histone H3 antibody, so that the final concentration of Sulfo-SMCC is 1.0-2.0 mM. The reaction system is incubated at room temperature for 30-40 min with gentle shaking, so that the NHS ester group of Sulfo-SMCC reacts with the surface of the antibody molecule to form a stable amide bond. After the reaction is completed, the reaction solution is subjected to ultrafiltration treatment to remove unreacted Sulfo-SMCC and its by-products, and a maleimide-modified anti-histone H3 antibody is obtained.

[0063] The glucose oxidase is dissolved in PBS buffer, and an aqueous solution of Traut's reagent is added to the glucose oxidase, so that the final concentration of Traut's reagent is 100-200 μM. The reaction system is incubated at room temperature (25±2°C) for 30-40 min in the dark, so that free thiol groups (-SH) are introduced into the glucose oxidase to form thiolated glucose oxidase. After the reaction is completed, the reaction solution is also subjected to ultrafiltration treatment to remove unreacted substances and their by-products, and a thiol-modified glucose oxidase is obtained.

[0064] The maleimide-modified anti-histone H3 antibody and the thiol-modified glucose oxidase are mixed at a molar ratio of antibody: enzyme of 1:3-1:5, and the mixture is incubated at room temperature in the dark for 1-2 h with gentle shaking, so that a stable thioether bond is formed between the antibody and the enzyme to achieve covalent coupling. After the coupling reaction is completed, a cysteine solution with a final concentration of 5 mM (blocking agent) is added to the system for further reaction for 20-30 min, and the reaction solution is then purified to obtain the histone H3 antibody-glucose oxidase conjugate.

[0065] The preparation method of the MPO antibody-horseradish peroxidase conjugate is as follows:

[0066] The MPO antibody was dissolved in PBS buffer, and a sulfonyl-maleimide methylcyclohexanecarboxylic acid succinimide ester solution was added to bring the final concentration of Sulfo-SMCC to 1.0-2.0 mM. The reaction system was gently incubated at room temperature for 30-40 min with shaking to allow the NHS ester groups of Sulfo-SMCC to undergo an amidation reaction with the amino groups of lysine residues on the surface of the antibody molecule, introducing active maleimide groups onto the antibody. After the reaction was completed, the reaction solution was ultrafiltered to completely remove unreacted Sulfo-SMCC and its byproducts, obtaining the maleimide-modified MPO antibody.

[0067] Horseradish peroxidase (HRP) was dissolved in PBS buffer, and an aqueous solution of Traut's reagent was added to bring the final concentration of Traut's reagent to 100-200 μM. The reaction system was incubated at room temperature in the dark for 30-40 min to allow the Traut's reagent to react with the lysine residues on the surface of HRP, forming thiolated horseradish peroxidase. After the reaction was complete, ultrafiltration was performed to obtain the thiolated horseradish peroxidase.

[0068] Maleimide-modified MPO antibody and thiolated horseradish peroxidase were mixed at an antibody:enzyme molar ratio of 1:3-1:5. The mixture was incubated at room temperature in the dark for 1-2 hours, with gentle shaking during incubation to allow stable thioether bonds to form between the antibody and enzyme, achieving covalent coupling. After the coupling reaction was complete, a 5 mM cysteine ​​solution was added to the system as a blocking agent, and the reaction was continued for 20-30 minutes. Finally, the reaction solution was purified by ultrafiltration to obtain the MPO antibody-horseradish peroxidase conjugate.

[0069] The preparation method of the double-stranded DNA aptamer-alkaline phosphatase conjugate is as follows:

[0070] Double-stranded DNA aptamers were dissolved in PBS buffer, and sulfonyl-maleimide methylcyclohexanecarboxylic acid succinimide ester solution was added to bring the final concentration of Sulfo-SMCC to 1.0-2.0 mM. The reaction system was gently incubated at room temperature for 30-40 min with gentle shaking to allow the formation of stable amide bonds between Sulfo-SMCC and the aptamer, thereby introducing active maleimide groups onto the aptamer. After the reaction was completed, ultrafiltration was performed to obtain maleimide-modified double-stranded DNA aptamers.

[0071] The alkaline phosphatase (ALP) is dissolved in PBS buffer, and an aqueous solution of Traut's reagent is added to the solution, so that the final concentration of Traut's reagent is 100-200 μM. The reaction system is incubated at room temperature for 30-40 min in the dark, so that the thiolated alkaline phosphatase is formed between Traut's reagent and ALP molecules. After the reaction is completed, ultrafiltration treatment is performed to obtain the thiolated modified alkaline phosphatase.

[0072] The maleimide-modified double-stranded DNA aptamer is mixed with the thiolated alkaline phosphatase at a molar ratio of 1:3-1:5 of aptamer: enzyme, and the mixture is incubated at room temperature for 1-2 h in the dark, with gentle shaking, so that a stable thioether bond is formed between the aptamer and the enzyme, and the covalent coupling is completed. After the coupling reaction is completed, a cysteine solution with a final concentration of 5 mM is added to the system, and the reaction is continued for 20-30 min. Finally, after ultrafiltration treatment, the double-stranded DNA aptamer-alkaline phosphatase conjugate is obtained.

[0073] In order to further clarify the scheme of the present application and its technical progress, the following specific examples and technical effects are described.

[0074] Example 1

[0075] The present embodiment provides a preparation method of a multi-modal nano probe NETs detection kit, which comprises the following steps:

[0076] S1: Disperse the gold nanostar with an average particle size of 80 nm in a mercaptoundecanoic acid solution with a concentration of 0.15 mM, and then add mercapto polyethylene glycol with a molecular weight of 11 kDa after oscillation treatment. React at 4℃ for 14 h to obtain a first mixture.

[0077] S2: Add PBS buffer to the first mixture, and then sequentially add the histone H3 antibody-glucose oxidase conjugate, the MPO antibody-horseradish peroxidase conjugate, and the double-stranded DNA aptamer-alkaline phosphatase conjugate. The molar ratio of the histone H3 antibody, the MPO antibody, and the double-stranded DNA aptamer is 1:1:1. Perform incubation treatment.

[0078] S3: After the incubation treatment is completed, add 4-(N-maleimido methyl) cyclohexane-1-carboxylic acid sulfonic succinimidyl ester sodium salt to the system, and react at 4℃ for 35 min. Remove the free components after the reaction. Add PBS buffer to make the final concentration of the gold nanostar 4.5 nM to obtain the nano probe.

[0079] Provide Tris-HCl buffer with a pH of 8.8 as the chemiluminescence working solution, wherein the concentration of glucose is 8 mM, the concentration of luminol is 0.2 mM, the concentration of p-nitrophenyl phosphate is 1.5 mM, and the mass concentration of p-iodophenol is 0.15%.

[0080] The preparation method of the histone H3 antibody-glucose oxidase conjugate is as follows:

[0081] The anti-histone H3 antibody is dissolved in PBS buffer, and a sulfo-maleimide methylcyclohexane carboxylic acid succinimidyl ester solution is added to the anti-histone H3 antibody to obtain a final concentration of 1.5 mM of Sulfo-SMCC. The reaction system is incubated at room temperature for 35 min with gentle shaking, and then ultrafiltration is performed to obtain the maleimide-modified anti-histone H3 antibody. The glucose oxidase is dissolved in PBS buffer, and an aqueous solution of Traut's reagent is added to the glucose oxidase to obtain a final concentration of 150 μM of Traut's reagent. The reaction system is incubated at room temperature for 35 min in the dark, and after the reaction is completed, ultrafiltration is performed to obtain the thiol-modified glucose oxidase. The maleimide-modified anti-histone H3 antibody and the thiol-modified glucose oxidase are mixed at a molar ratio of 1:4, and the mixture is incubated at room temperature for 1.5 h in the dark with gentle shaking. After the reaction is completed, a cysteine solution with a final concentration of 5 mM is added for a further reaction for 25 min, and after the impurities are removed, the histone H3 antibody-glucose oxidase conjugate is obtained.

[0082] The preparation method of the MPO antibody-horseradish peroxidase conjugate is as follows:

[0083] The MPO antibody is dissolved in PBS buffer, and a sulfo-maleimide methylcyclohexane carboxylic acid succinimidyl ester solution is added to the MPO antibody to obtain a final concentration of 1.5 mM of Sulfo-SMCC. The reaction system is incubated at room temperature for 30-40 min with gentle shaking, and then ultrafiltration is performed to obtain the maleimide-modified MPO antibody. The horseradish peroxidase is dissolved in PBS buffer, and an aqueous solution of Traut's reagent is added to the horseradish peroxidase to obtain a final concentration of 150 μM of Traut's reagent. The reaction system is incubated at room temperature for 30-40 min in the dark, and then ultrafiltration is performed to obtain the thiol-modified horseradish peroxidase. The maleimide-modified MPO antibody and the thiol-modified horseradish peroxidase are mixed at a molar ratio of 1:4, and the mixture is incubated at room temperature for 1.5 h in the dark with gentle shaking. After the reaction is completed, a cysteine solution with a final concentration of 5 mM is added for a further reaction for 25 min, and after ultrafiltration, the MPO antibody-horseradish peroxidase conjugate is obtained.

[0084] The preparation method of the double-stranded DNA aptamer-alanine phosphatase conjugate is as follows:

[0085] The double-stranded DNA aptamer is dissolved in PBS buffer, and a sulfo-maleimide methylcyclohexane carboxylic acid succinimidyl ester solution is added to the double-stranded DNA aptamer to make the final concentration of Sulfo-SMCC 1.5 mM. The reaction system is incubated at room temperature for 35 min with gentle shaking, and then ultrafiltration is performed to obtain the maleimide-modified double-stranded DNA aptamer. The alkaline phosphatase is dissolved in PBS buffer, and an aqueous solution of Traut's reagent is added to the alkaline phosphatase to make the final concentration of Traut's reagent 150 μM. The reaction system is incubated at room temperature for 35 min in the dark, followed by ultrafiltration treatment to obtain the thiolated modified alkaline phosphatase. The maleimide-modified double-stranded DNA aptamer is mixed with the thiolated alkaline phosphatase at a molar ratio of 1:4, and the mixture is incubated at room temperature for 1.5 h in the dark with gentle shaking. After the reaction is completed, a cysteine solution with a final concentration of 5 mM is added to the system, and the reaction is continued for 25 min. Then, ultrafiltration treatment is performed to obtain the double-stranded DNA aptamer-alkaline phosphatase conjugate.

[0086] Example 2

[0087] The present embodiment provides a preparation method of a multi-modal nano probe NETs detection kit, comprising the following steps:

[0088] S1: Gold nanostars with an average particle size of 70 nm are dispersed in a mercaptoundecanoic acid solution with a concentration of 0.1 mM. After oscillation treatment, thiolated polyethylene glycol with a molecular weight of 10 kDa is added, and the reaction is carried out at 4°C for 12 h to obtain a first mixture.

[0089] S2: PBS buffer is added to the first mixture, and then a histone H3 antibody-glucose oxidase conjugate, an MPO antibody-horseradish peroxidase conjugate, and a double-stranded DNA aptamer-alkaline phosphatase conjugate are sequentially added. The molar ratio of the histone H3 antibody, the MPO antibody, and the double-stranded DNA aptamer is 1:1:1, and incubation treatment is performed.

[0090] S3: After the incubation treatment is completed, 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt is added to the system, and the reaction is carried out at 4°C for 30 min. After the reaction, the free components are removed, PBS buffer is added to make the final concentration of the gold nanostars 4 nM, and a nano probe is obtained.

[0091] A Tris-HCl buffer with a pH of 8.5 is provided as a chemiluminescence working solution, wherein the concentration of glucose is 5 mM, the concentration of luminol is 0.1 mM, the concentration of p-nitrophenyl phosphate is 1.0 mM, and the mass concentration of p-iodophenol is 0.1%.

[0092] The preparation method of the histone H3 antibody-glucose oxidase conjugate is as follows:

[0093] The anti-histone H3 antibody was dissolved in PBS buffer, and a sulfo-maleimide methyl cyclohexane carboxylic acid succinimidyl ester solution was added to the anti-histone H3 antibody to obtain a final concentration of 1.0 mM of Sulfo-SMCC. The reaction system was incubated at room temperature for 30 min with gentle shaking, and then ultrafiltration was performed to obtain the maleimide-modified anti-histone H3 antibody. The glucose oxidase was dissolved in PBS buffer, and an aqueous solution of Traut's reagent was added to the glucose oxidase to obtain a final concentration of 100 μM of Traut's reagent. The reaction system was incubated at room temperature for 30 min in the dark, and after the reaction was completed, ultrafiltration was performed to obtain the thiol-modified glucose oxidase. The maleimide-modified anti-histone H3 antibody and the thiol-modified glucose oxidase were mixed at a molar ratio of 1:3, and the mixture was incubated at room temperature for 1 h in the dark with gentle shaking. After the reaction was completed, a cysteine solution was added to obtain a final concentration of 5 mM, and the reaction was continued for 20 min. After the impurities were removed, the histone H3 antibody-glucose oxidase conjugate was obtained.

[0094] The preparation method of the MPO antibody-horseradish peroxidase conjugate is as follows:

[0095] The MPO antibody was dissolved in PBS buffer, and a sulfo-maleimide methyl cyclohexane carboxylic acid succinimidyl ester solution was added to the MPO antibody to obtain a final concentration of 1.0 mM of Sulfo-SMCC. The reaction system was incubated at room temperature for 30 min with gentle shaking, and then ultrafiltration was performed to obtain the maleimide-modified MPO antibody. The horseradish peroxidase was dissolved in PBS buffer, and an aqueous solution of Traut's reagent was added to the horseradish peroxidase to obtain a final concentration of 100 μM of Traut's reagent. The reaction system was incubated at room temperature for 30 min in the dark, and then ultrafiltration was performed to obtain the thiol-modified horseradish peroxidase. The maleimide-modified MPO antibody and the thiol-modified horseradish peroxidase were mixed at a molar ratio of 1:3-1:5, and the mixture was incubated at room temperature for 1 h in the dark with gentle shaking. After the reaction was completed, a cysteine solution was added to obtain a final concentration of 5 mM, and the reaction was continued for 20 min. After ultrafiltration, the MPO antibody-horseradish peroxidase conjugate was obtained.

[0096] The preparation method of the double-stranded DNA aptamer-alkaline phosphatase conjugate is as follows:

[0097] The double-stranded DNA aptamer is dissolved in PBS buffer, and a sulfo-maleimide methylcyclohexane carboxylic acid succinimidyl ester solution is added to the double-stranded DNA aptamer to make the final concentration of Sulfo-SMCC 1.0 mM. The reaction system is incubated at room temperature for 30 min with gentle shaking, and then ultrafiltration is performed to obtain the maleimide-modified double-stranded DNA aptamer. The alkaline phosphatase is dissolved in PBS buffer, and an aqueous solution of Traut's reagent is added to the alkaline phosphatase to make the final concentration of Traut's reagent 100 μM. The reaction system is incubated at room temperature for 30 min in the dark, followed by ultrafiltration treatment to obtain the thiolated alkaline phosphatase. The maleimide-modified double-stranded DNA aptamer is mixed with the thiolated alkaline phosphatase at a molar ratio of 1:3, and the mixture is incubated at room temperature for 1 h in the dark with gentle shaking. After the reaction is completed, a cysteine solution with a final concentration of 5 mM is added to the system, and the reaction is continued for 20 min. Then, ultrafiltration treatment is performed to obtain the double-stranded DNA aptamer-alkaline phosphatase conjugate.

[0098] Example 3

[0099] The present embodiment provides a preparation method of a multi-modal nano probe NETs detection kit, comprising the following steps:

[0100] S1: Gold nanostars with an average particle size of 90 nm are dispersed in a mercaptoundecanoic acid solution with a concentration of 0.2 mM, and after oscillation treatment, mercapto polyethylene glycol with a molecular weight of 12 kDa is added. The reaction is carried out at 4°C for 16 h to obtain a first mixture.

[0101] S2: PBS buffer is added to the first mixture, and then a histone H3 antibody-glucose oxidase conjugate, an MPO antibody-horseradish peroxidase conjugate, and a double-stranded DNA aptamer-alkaline phosphatase conjugate are sequentially added. The molar ratio of the histone H3 antibody, the MPO antibody, and the double-stranded DNA aptamer is 1:1:1, and incubation treatment is performed.

[0102] S3: After the incubation treatment is completed, 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt is added to the system, and the reaction is carried out at 4°C for 40 min. After the reaction, the free components are removed, PBS buffer is added to make the final concentration of the gold nanostars 5 nM, and a nano probe is obtained.

[0103] A Tris-HCl buffer with a pH of 9.0 is provided as a chemiluminescence working solution, in which the concentration of glucose is 10 mM, the concentration of luminol is 0.3 mM, the concentration of p-nitrophenyl phosphate is 2.0 mM, and the mass concentration of p-iodophenol is 0.2%.

[0104] The preparation method of the histone H3 antibody-glucose oxidase conjugate is as follows:

[0105] The anti-histone H3 antibody was dissolved in PBS buffer, and a sulfo-maleimide methyl cyclohexane carboxylic acid succinimidyl ester solution was added to the anti-histone H3 antibody to obtain a final concentration of 2.0 mM of Sulfo-SMCC. The reaction system was incubated at room temperature for 30-40 min with gentle shaking, and then ultrafiltration was performed to obtain the maleimide-modified anti-histone H3 antibody. The glucose oxidase was dissolved in PBS buffer, and an aqueous solution of Traut's reagent was added to the glucose oxidase to obtain a final concentration of 200 μM of Traut's reagent. The reaction system was incubated at room temperature for 40 min in the dark, and after the reaction was completed, ultrafiltration was performed to obtain the thiol-modified glucose oxidase. The maleimide-modified anti-histone H3 antibody and the thiol-modified glucose oxidase were mixed at a molar ratio of 1:5, and the mixture was incubated at room temperature for 2 h in the dark with gentle shaking. After the reaction was completed, a cysteine solution was added to obtain a final concentration of 5 mM, and the reaction was continued for 30 min. After the impurities were removed, the histone H3 antibody-glucose oxidase conjugate was obtained.

[0106] The preparation method of the MPO antibody-horseradish peroxidase conjugate is as follows:

[0107] The MPO antibody was dissolved in PBS buffer, and a sulfo-maleimide methyl cyclohexane carboxylic acid succinimidyl ester solution was added to the MPO antibody to obtain a final concentration of 2.0 mM of Sulfo-SMCC. The reaction system was incubated at room temperature for 40 min with gentle shaking, and then ultrafiltration was performed to obtain the maleimide-modified MPO antibody. The horseradish peroxidase was dissolved in PBS buffer, and an aqueous solution of Traut's reagent was added to the horseradish peroxidase to obtain a final concentration of 200 μM of Traut's reagent. The reaction system was incubated at room temperature for 40 min in the dark, and then ultrafiltration was performed to obtain the thiol-modified horseradish peroxidase. The maleimide-modified MPO antibody and the thiol-modified horseradish peroxidase were mixed at a molar ratio of 1:5, and the mixture was incubated at room temperature for h in the dark with gentle shaking. After the reaction was completed, a cysteine solution was added to obtain a final concentration of 5 mM, and the reaction was continued for 30 min. After ultrafiltration, the MPO antibody-horseradish peroxidase conjugate was obtained.

[0108] The preparation method of the double-stranded DNA aptamer-alkaline phosphatase conjugate is as follows:

[0109] The double-stranded DNA aptamer was dissolved in PBS buffer, and a sulfo-maleimide methylcyclohexane carboxylic acid succinimidyl ester solution was added to the solution to make the final concentration of Sulfo-SMCC 2.0 mM. The reaction system was incubated at room temperature for 40 min with gentle shaking, and then ultrafiltration was performed to obtain the maleimide-modified double-stranded DNA aptamer. The alkaline phosphatase was dissolved in PBS buffer, and an aqueous solution of Traut's reagent was added to the solution to make the final concentration of Traut's reagent 200 μM. The reaction system was incubated at room temperature for 40 min in the dark, followed by ultrafiltration treatment to obtain the thiolated modified alkaline phosphatase. The maleimide double-stranded DNA aptamer was mixed with the thiolated alkaline phosphatase at a molar ratio of 1:5, and the mixture was incubated at room temperature in the dark for 2 h with gentle shaking. After the reaction was completed, a cysteine solution with a final concentration of 5 mM was added to the system, and the reaction was continued for 30 min. Then, ultrafiltration treatment was performed to obtain the double-stranded DNA aptamer-alkaline phosphatase conjugate.

[0110] Take the recombinant human NETs standard containing complete NETs (including dsDNA, histone H3, MPO), dilute it with PBS buffer containing 1wt% BSA to a gradient concentration, and obtain NETs standard solutions with concentrations of 0 nM, 1.5 nM, 5 nM, 15 nM, 30 nM, 50 nM, and 100 nM, respectively. Take 100 μL of each concentration of NETs standard solution, add an equal volume of nanoprobes solution (keeping the gold nanostar concentration the same), and incubate at 37°C for 18 min to form probe-NETs complexes. Continue to add 100 μL of 9wt% PEG solution, vortex well, and then centrifuge at 3000 rpm for 10 min, discard the supernatant and retain the precipitate. Add an equal volume of chemiluminescence working solution to the precipitate, and immediately after 6 min of reaction at 37°C in the dark, detect the relative luminescence intensity (RLU value) using a chemiluminescence instrument.

[0111] Take the NETs concentration as the abscissa and the corresponding RLU value as the ordinate to establish a standard curve of relative luminescence intensity versus NETs concentration, as shown in Figure 1 The linear correlation coefficient of relative luminescence intensity and NETs concentration is >0.999.

[0112] A series of standard solutions with known concentrations were prepared using recombinant human NETs, with concentrations of 0 nM, 0.02 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, and 5.0 nM, respectively, dissolved in healthy human serum matrix to simulate the real sample environment. Take 100 μL of each concentration of standard sample, and use the kit prepared in Examples 1-3 to perform the following detection:

[0113] A1: The same volume of standard sample was added into the nano-probes prepared in Examples 1-3, respectively, and the probe-NETs complex was obtained after incubation at 37°C for 15-20 min.

[0114] A2: The same volume of polyethylene glycol solution with a concentration of 8-10 wt% was added into the probe-NETs complex, and the precipitate was obtained after centrifugal treatment after mixing and discarding the supernatant.

[0115] A3: The chemiluminescence working solution of Examples 1-3 was added into the different precipitates, respectively, and the relative luminescence intensity of the detection system was detected after dark reaction at 37°C for 5-8 min, and the concentration of NETs was calculated, which is shown in Table 1.

[0116]

[0117] As shown in Table 1, the detection range of the kit of Examples 1-3 is 0-5.0 nM, and the detection results of lower content of NETs are relatively accurate, which has higher sensitivity and higher accuracy, and has good repeatability and stability.

[0118] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A multimodal nanoprobe (NET) detection kit, characterized in that, The invention includes nanoprobes and chemiluminescent working solutions. The nanoprobes include gold nanostars, histone H3 antibody-glucose oxidase conjugates, MPO antibody-horseradish peroxidase conjugates, and double-stranded DNA aptamers-alkaline phosphatase conjugates immobilized on the surface of gold nanostars. Histone H3 antibody-glucose oxidase conjugate is a complex formed by chemical cross-linking of anti-histone H3 antibody and glucose oxidase; MPO antibody-horseradish peroxidase conjugate is a complex formed by chemical cross-linking of MPO antibody and horseradish peroxidase; and double-stranded DNA aptamer-alkaline phosphatase conjugate is a complex formed by chemical cross-linking of double-stranded DNA aptamer and alkaline phosphatase. The chemiluminescent working solution is a Tris-HCl buffer containing glucose, luminol, p-nitrophenyl phosphate, and an enhancer.

2. The multimodal nanoprobe NETs detection kit according to claim 1, characterized in that, In the chemiluminescence working solution, the enhancer is p-iodophenol, with a mass concentration of 0.1-0.2%, the concentration of glucose is 5-10 mM, the concentration of luminol is 0.1-0.3 mM, the concentration of p-nitrophenyl phosphate is 1.0-2.0 mM, and the pH of the Tris-HCl buffer is 8.5-9.

0.

3. The multimodal nanoprobe NETs detection kit according to claim 1, characterized in that, The particle size of gold nanostars is 70-90 nm; In the histone H3 antibody-glucose oxidase conjugate, the molar ratio of histone H3 antibody to glucose oxidase is 1:3-1:5; In the MPO antibody-horseradish peroxidase conjugate, the molar ratio of MPO antibody to horseradish peroxidase is 1:3-1:

5. In the double-stranded DNA aptamer-alkaline phosphatase conjugate, the molar ratio of double-stranded DNA aptamer to alkaline phosphatase is 1:3 to 1:

5.

4. A method for preparing a multimodal nanoprobe (NET) detection kit according to any one of claims 1-3, characterized in that, Includes the following steps: S1: Disperse gold nanostars in mercaptoundecanoic acid solution, shake and add mercapto polyethylene glycol, and after reaction, obtain the first mixture; S2: Add PBS buffer to the first mixture, then add histone H3 antibody-glucose oxidase conjugate, MPO antibody-horseradish peroxidase conjugate and double-stranded DNA aptamer-alkaline phosphatase conjugate in sequence, and incubate. S3: After the incubation treatment is completed, sodium salt of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester is added to the system, and the nano probe is obtained after the reaction.

5. The preparation method of the multimodal nanoprobe NETs detection kit according to claim 4, characterized in that, In step S1, the concentration of the mercaptoundecanoic acid solution is 0.1-0.2 mM, and after adding mercapto polyethylene glycol, the reaction is carried out at 4°C for 12-16 h. The molecular weight of mercapto polyethylene glycol is 10-12 kDa.

6. The preparation method of the multimodal nanoprobe NETs detection kit according to claim 4, characterized in that, In step S2, the molar ratio of histone H3 antibody, MPO antibody, and double-stranded DNA aptamer is 1:1:1; in step S3, after adding sodium salt of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester, the reaction is carried out at 4°C for 30-40 min.

7. A method for detecting NETs concentration using the multimodal nanoprobe NETs detection kit according to any one of claims 1-3 or the multimodal nanoprobe NETs detection kit prepared by the preparation method according to any one of claims 4-6, wherein the method is for non-disease diagnostic purposes, characterized in that, Includes the following steps: A1: The sample to be tested is added to the nanoprobe and incubated to obtain the probe-NETs complex; A2: Add an equal volume of polyethylene glycol solution to the probe-NETs complex, mix well, centrifuge, discard the supernatant, and obtain the precipitate; A3: Add chemiluminescent working solution to the precipitate, react in the dark, and then detect the relative luminescence intensity of the system to calculate the concentration of NETs.

8. The method for detecting NETs concentration according to claim 7, characterized in that, In step A1, the incubation temperature is 37℃ and the incubation time is 15-20 minutes.

9. The method for detecting NETs concentration according to claim 7, characterized in that, In step A2, the concentration of the polyethylene glycol solution is 8-10 wt%.

10. The method for detecting NETs concentration according to claim 7, characterized in that, In step A3, the chemiluminescent working solution and the probe-NETs complex have the same volume, the reaction temperature is 37℃, and the reaction time is 5-8 min.

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