CN112881680B

CN112881680B - Method for preparing dsDNA-linked magnetic particles

Info

Publication number: CN112881680B
Application number: CN202110066358.4A
Authority: CN
Inventors: 祝亮; 谭松暖; 何凡; 钱纯亘
Original assignee: Shenzhen Zhuoren Biotechnology Co ltd
Current assignee: Shenzhen Zhuoren Biotechnology Co ltd
Priority date: 2021-01-19
Filing date: 2021-01-19
Publication date: 2022-11-15
Anticipated expiration: 2041-01-19
Also published as: CN112881680A

Abstract

The invention relates to a preparation method of magnetic particles connected with dsDNA, which comprises the following steps: activating carboxyl groups of the carboxyl magnetic particles to prepare activated carboxyl magnetic particles; preheating the dsDNA for 0.5-3 h at 25-60 ℃ to prepare preheated dsDNA; and preparing the magnetic particles connected with the dsDNA by mixing and reacting the 4-dimethylamino pyridine, the activated carboxyl magnetic particles and the preheated dsDNA at the temperature of 25-60 ℃. The preparation method has high efficiency and low cost, and the prepared magnetic particles connected with the dsDNA have good specificity.

Description

Method for preparing dsDNA-linked magnetic particles

Technical Field

The invention relates to the field of biotechnology, in particular to a preparation method of magnetic particles connected with dsDNA.

Background

A double stranded DNA (dsDNA) antibody is an autoantibody capable of binding to human DNA. dsDNA antibodies are present in the serum of more than 90% of patients with Systemic Lupus Erythematosus (SLE). The determination of dsDNA antibodies is of great significance for the diagnosis and differential diagnosis of SLE, monitoring treatment and disease tracking, and prognosis judgment, etc.

The main methods for detecting the dsDNA antibody include enzyme-linked immunosorbent assay, chemiluminescence immunoassay, immunofluorescence assay and the like, wherein the chemiluminescence immunoassay has the advantages of high sensitivity, wide linear range, rapid and simple detection, stable result and the like, so that the development is faster and faster. However, chemiluminescent immunoassays require magnetic particles with dsDNA attached. At present, the magnetic particles connected with the dsDNA are mainly formed by combining magnetic beads labeled by avidin with dsDNA labeled by biotin. Wherein, the preparation method of the biotin-labeled dsDNA mainly comprises a PCR amplification method and a photosensitive biotin labeling method. The PCR amplification method needs enzyme, dNTP and biotin-labeled dUTP, so that the production cost is high and the industrial popularization is difficult; in the light-sensitive biotin labeling method, light-sensitive biotin is randomly inserted into a base of DNA, so that the specificity of the combination of the DNA and an antibody thereof is easily reduced. Thus, current methods for preparing dsDNA-linked magnetic beads are either costly or less specific.

Disclosure of Invention

Based on this, there is a need for a method for preparing biotin-labeled dsDNA at a lower cost and with good specificity.

A method of preparing dsDNA-linked magnetic microparticles comprising the steps of:

activating the carboxyl group of the carboxyl group magnetic particles to prepare activated carboxyl group magnetic particles;

preheating the solution containing the dsDNA for 0.5 to 3 hours at the temperature of between 25 and 60 ℃ to prepare preheated dsDNA; and

and mixing and reacting the 4-dimethylamino pyridine, the activated carboxyl magnetic particles and the preheated dsDNA at the temperature of 25-60 ℃ to prepare the magnetic particles connected with the dsDNA.

The preparation method of the magnetic particles connected with the dsDNA has the advantages that the hydroxyl at the 3' tail end of the dsDNA is more fully exposed through heating, the reaction activity of the dsDNA is improved, 4-dimethylaminopyridine is added for catalysis, so that the esterification reaction of the dsDNA and the carboxyl magnetic particles is rapidly carried out, the efficiency of preparing the magnetic particles connected with the dsDNA is improved, and the preparation method does not need enzyme, is simple and easy to operate and saves the cost. Meanwhile, because the photosensitive label is not introduced in a random insertion mode, and the dsDNA is heated at the temperature of 25-60 ℃ without causing irreversible change of the structure of the dsDNA, the specificity of the combination of the dsDNA and the antibody thereof is not influenced. Therefore, the preparation method has high efficiency and low cost, and the prepared magnetic particles connected with the dsDNA have good specificity.

In one embodiment, the pre-heat treatment temperature of the dsDNA-containing solution is 35 ℃ to 50 ℃.

In one embodiment, the time for heat treatment of the dsDNA containing solution is 1h to 2h.

In one embodiment, in the step of mixing and reacting the 4-dimethylaminopyridine, the activated carboxyl magnetic particles and the preheated dsDNA, the temperature of the mixing and reacting is 35-50 ℃.

In one embodiment, the molar ratio of 4-dimethylaminopyridine to carboxyl groups in the activated carboxyl magnetic particles is (0.05-0.3): 1.

in one embodiment, in the step of mixing and reacting 4-dimethylaminopyridine, the activated carboxyl magnetic particles and the preheated dsDNA, the pH of a mixed solution of the activated carboxyl magnetic particles and the preheated dsDNA is 6-7.5.

In one embodiment, the carboxyl magnetic particles are carboxyl nano magnetic beads, and the particle size of the carboxyl magnetic particles is 0.5-3 μm.

In one embodiment, in the step of mixing and reacting 4-dimethylaminopyridine, the activated carboxyl magnetic particles and the preheated dsDNA, the mass of the preheated dsDNA is 0.05-2 times of the mass of the activated carboxyl magnetic particles.

In one embodiment, the dsDNA containing solution is prepared by dissolving dsDNA in a buffer solution, the pH of the dsDNA containing solution is 7-8.5, and the buffer solution does not contain amino, hydroxyl and sulfhydryl.

In one embodiment, the buffer is selected from at least one of a phosphate buffer, a carbonate buffer, and a borate buffer.

In one embodiment, the carboxyl groups of the carboxyl magnetic particles are activated with carbodiimide and hydroxysuccinimide.

Drawings

FIG. 1 is a reaction scheme of example 1;

figure 2 is a linear fit plot of dsDNA antibody concentration versus luminescence in test two.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

One embodiment of the present invention provides a method for preparing magnetic particles having dsDNA attached thereto, the method comprising steps a to c. Specifically, the method comprises the following steps:

step a: the carboxyl groups of the carboxyl group magnetic particles are activated to prepare activated carboxyl group magnetic particles.

Specifically, the carboxyl magnetic particles are carboxyl nano magnetic beads. Further, the particle size of the carboxyl magnetic fine particles is 0.5 to 3 μm. Of course, in other embodiments, the shape of the carboxyl magnetic fine particles is not limited to beads, and may be other shapes. The particle size of the carboxyl magnetic fine particles is not limited to 0.5 to 3 μm, and may be other sizes. The particle size herein is a median particle size (D50).

Specifically, the carboxyl group of the carboxyl magnetic fine particle is activated with carbodiimide and hydroxysuccinimide. Optionally, the carbodiimide is selected from at least one of dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N, N' -diisopropylcarbodiimide; the hydroxysuccinimide is selected from at least one of N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide (Sulfo-NHS). Further, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide were used to activate the carboxyl groups of the carboxyl magnetic fine particles.

In one embodiment, the molar ratio of the carbodiimide to the carboxyl group of the carboxyl group magnetic fine particle is (1 to 10): 1. alternatively, the molar ratio of carbodiimide to carboxyl groups of the carboxyl group magnetic fine particles is 1: 1. 3: 1. 5: 1. 7:1 or 8:1. further, the molar ratio of the carbodiimide to the carboxyl group of the carboxyl group magnetic fine particles is (1 to 4): 1.

in one embodiment, the molar ratio of the hydroxysuccinimide to the carboxyl groups of the carboxyl-based magnetic particles is (0.1 to 5): 1. further, the molar ratio of the hydroxysuccinimide to the carboxyl group of the carboxyl group magnetic fine particles is (1 to 3): 1.

in one embodiment, the temperature of activation is 4 ℃ to 30 ℃; the activation time is 0.5 h-4 h. Further, the activation temperature is 10-30 ℃; the activation time is 0.5 h-2 h.

Further, the carboxyl groups of the activated carboxyl magnetic fine particles were carried out in a 2- (N-morpholino) ethanesulfonic acid buffer solution having a pH of 4 to 7.

It is understood that, in other embodiments, the way of activating the carboxyl groups of the carboxyl magnetic particles is not limited to the way of using carbodiimide and hydroxysuccinimide, but also can be other ways as long as the carboxyl groups of the carboxyl magnetic particles can be activated to perform esterification reaction with the hydroxyl groups of the dsDNA.

Step b: preheating the solution containing the dsDNA for 0.5 to 3 hours at the temperature of between 25 and 60 ℃ to prepare the preheated dsDNA.

The dsDNA has low reactivity due to the influence of the helical structure, and the reaction efficiency is low when the carboxyl of the activated magnetic particles and the hydroxyl on the 3' end of the dsDNA are directly subjected to esterification reaction. The preparation method of the dsDNA-connected magnetic particles enhances the reaction activity of the dsDNA by heating the dsDNA, so that the reaction efficiency is improved, and simultaneously, the problems of higher preparation cost or poorer specificity caused by using the biotin-labeled dsDNA are avoided.

Alternatively, the temperature of the heat treatment is 25 ℃, 30 ℃, 35 ℃,37 ℃, 38 ℃, 40 ℃, 42 ℃, 45 ℃, 50 ℃, 53 ℃, 55 ℃, 58 ℃ or 60 ℃. Further, the temperature of the heat treatment is 35 to 50 ℃.

Optionally, the time of the heat treatment is 0.5h, 0.8h, 1h, 1.5h, 2h, 2.5h or 3h. Further, the time of the heat treatment is 1 to 2 hours.

In one embodiment, the step of preparing the preheated dsDNA comprises: dissolving dsDNA in a buffer solution to prepare a dsDNA solution; and preheating the dsDNA solution at 25-60 ℃ for 0.5-3 h to prepare preheated dsDNA. Wherein the buffer solution does not contain amino, hydroxyl and sulfydryl; further, the pH of the buffer solution for preparing the dsDNA solution is 7 to 8.5. Optionally, the buffer to formulate the dsDNA solution is selected from at least one of a phosphate buffer, a carbonate buffer, and a borate buffer.

Step c: and mixing and reacting the 4-dimethylamino pyridine, the activated carboxyl magnetic particles and the preheated dsDNA at the temperature of 25-60 ℃ to prepare the magnetic particles connected with the dsDNA.

Specifically, under the action of 4-dimethylamino pyridine, the carboxyl of the activated carboxyl magnetic particles and the hydroxyl of the preheated dsDNA undergo esterification reaction rapidly to form ester bonds, so that the dsDNA is connected to the carboxyl magnetic particles. Further, the temperature of the esterification reaction is 25-60 ℃; the time of the esterification reaction is 2 to 30 hours. Furthermore, the temperature of the esterification reaction is 35-50 ℃; the esterification reaction time is 12-24 h.

Optionally, the mass of the preheated dsDNA is 0.05 to 2 times the mass of the activated carboxyl magnetic particles. Further, the mass of the preheated dsDNA is 0.5 to 2 times of the mass of the activated carboxyl magnetic particles.

Optionally, the molar ratio of the 4-dimethylaminopyridine to the carboxyl groups in the activated carboxyl magnetic particles is (0.05-0.3): 1. optionally, the pH of the mixed solution of the 4-dimethylaminopyridine, the activated carboxyl magnetic particles and the preheated dsDNA is 6-7.5.

Of course, after the mixing reaction of the activated carboxyl magnetic particles and the preheated dsDNA is finished, the method further comprises a step of washing the magnetic particles after the reaction and a step of storing the washed magnetic particles in a storage solution.

In one embodiment, the magnetic particles after the reaction are washed with a buffer. The buffer solution for washing the magnetic particles after the reaction is selected from at least one of a phosphate buffer solution, a carbonate buffer solution and a borate buffer solution.

In one embodiment, the preservation solution comprises a buffer solution that does not contain an amino group. Optionally, the preservation solution comprises at least one of phosphate buffered saline (PBS buffer) and 2- (N-morpholine) ethanesulfonic acid buffer (MES buffer). Further, saveThe liquid further comprises at least one of a preservative and a surfactant. Optionally, the preservative is selected from Na ₃ 5363 and Proclin 950, and N, proclin. Optionally, the surfactant is selected from at least one of tween, polyethylene glycol, triton and choline chloride.

In one embodiment, the pH of the preservation solution is between 5 and 8. Further, the pH of the storage solution is 6.5 to 7.5.

The preparation method of the dsDNA-linked magnetic particles has at least the following advantages:

(1) The above method for preparing dsDNA-attached magnetic particles allows the double helix structure of dsDNA to be opened and the reaction sites to be exposed by preheating dsDNA before the dsDNA is reacted with the activated magnetic particles, so that the reactivity of dsDNA is improved, and 4-dimethylaminopyridine can accelerate the reaction of the activated magnetic particles with dsDNA. Compared with the traditional method of firstly modifying the DNA structure (for example, introducing active groups such as amino, sulfydryl and the like) or labeling biotin and then reacting with the magnetic particles to connect the dsDNA to the magnetic particles, the preparation method of the magnetic particles connected with the dsDNA has low cost and good specificity.

(2) Although dsDNA can be connected to the magnetic particles by physical adsorption, this method is only suitable for DNA purification and separation, and does not meet the requirements of chemiluminescence immunoassay, and in the above method for preparing dsDNA-connected magnetic particles, dsDNA is connected to the magnetic particles by forming chemical bonds with the magnetic particles, and can be directly used for chemiluminescence immunoassay.

(3) The preparation method of the dsDNA-connected magnetic particle firstly activates the carboxyl of the magnetic particle and preheats the dsDNA, and then mixes the activated magnetic particle and the preheated dsDNA for reaction, so that the operation is simple, the efficiency of obtaining the dsDNA-connected magnetic particle is high, and the method is easy for industrial production.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures not specified for the specific conditions in the examples were carried out according to conventional conditions, such as those described in the literature, in books or as recommended by the manufacturer, hereinafter dsDNA is salmon sperm DNA from Sigma (cat. No. 31149-10G-F).

Example 1

Referring to fig. 1, the reagents used to prepare dsDNA-attached nanobeads in example 1 are shown in table 1, and the preparation method includes, but is not limited to, the following steps:

(1) 1mL of a carboxyl nanobead solution (the particle size of the carboxyl nanobead is 2 μm, the concentration of the carboxyl nanobead is 100mg/mL, and the carboxyl content is 600nmol/mg, that is, each 1mL of the carboxyl nanobead solution contains 100mg of the magnetic bead and each mg of the carboxyl nanobead contains 600nmol of the carboxyl group) is taken, treated in a magnetic separator for 3 minutes, removed from the supernatant by a pipettor, and washed 3 times with 10mL of 100mM MES buffer (pH 6).

(2) Adding 2mL of 50mM MES buffer solution (pH is 6) into the carboxyl nano magnetic beads washed in the step (1), then adding 3mL of EDC solution (the concentration of EDC is 10 mg/mL) and 3mL of Sulfo-NHS solution (the concentration of Sulfo-NHS is 8 mg/mL), and rotating at 25 ℃ (40 r/min) for reaction for 1h to obtain activated carboxyl nano magnetic beads.

(3) 10mL of dsDNA solution (dsDNA concentration 5 mg/mL) was prepared using 100mM phosphate buffer (pH 8), and then 10mL of dsDNA solution was bathed at 37 ℃ for 2 hours to obtain a preheated dsDNA solution.

(4) And (3) adding all the dsDNA preheated in the step (3) into the activated carboxyl nano magnetic beads in the step (2), controlling the pH of the mixed solution to be 7.2, adding 80 mu L of a DMSO solution of DMAP (the concentration of DMAP is 10 mg/mL), and rotating at 37 ℃ (60 revolutions per minute) for reaction for 24 hours to obtain a reaction solution containing the dsDNA-connected nano magnetic beads.

(5) And (4) performing magnetic separation on the reaction liquid containing the dsDNA-connected nano magnetic beads obtained in the step (4), removing a supernatant by using a pipette, and washing 3 times by 5mL of 50mM PBS buffer solution containing 0.1% (v/v) Tween-20, so as to obtain washed dsDNA-connected nano magnetic beads. The washed dsDNA-ligated nanobeads were stored in 10mL Tween-20 containing 0.1% (v/v)And 0.02% (m/v) Na ₃ N, 50mM phosphate buffer (pH 7.4) containing 0.2% (v/v) Proclin 300.

Example 2

The reagents used to prepare dsDNA-ligated nanobeads in example 2 are shown in table 1, and the preparation method includes, but is not limited to, the following steps:

(1) 1mL of a carboxyl nanobead solution (the particle size of the carboxyl nanobead is 0.5 μm, the concentration is 100mg/mL, and the carboxyl content is 200nmol/mg, that is, 100mg of the magnetic beads are contained in 1mL of the carboxyl nanobead solution, and 200nmol of carboxyl groups are contained in each mg of the carboxyl nanobead solution) is taken, treated in a magnetic separator for 3 minutes, removed from the supernatant by a pipette, and washed 3 times with 1mL of 100mM MES buffer (pH = 5.5).

(2) Adding 2mL of 50mM MES buffer solution (pH is 5.5) into the carboxyl nano magnetic beads washed in the step (1), then adding 1mL of EDC solution (EDC concentration is 10 mg/mL) and 1mL of Sulfo-NHS solution (Sulfo-NHS concentration is 8 mg/mL), and rotating at 20 ℃ (40 revolutions/min) for reaction for 1.5h to obtain activated carboxyl nano magnetic beads.

(3) A dsDNA solution was prepared at 10mL of 5mg/mL using a 100mM phosphate buffer solution (pH 8.2), and then, the solution at 10mL of 5mg/mL was subjected to a water bath at 45 ℃ for 1.5 hours to obtain a preheated dsDNA solution.

(4) And (3) adding all the preheated dsDNA in the step (3) into the activated carboxyl magnetic beads in the step (2), controlling the pH of the mixed solution to be 7.4, adding 30 mu L of a DMSO solution of DMAP (the concentration of DMAP is 10 mg/mL), and rotating at 45 ℃ (60 revolutions per minute) for reacting for 18 hours to obtain a reaction solution containing the nano magnetic beads connected with the dsDNA.

(5) And (3) performing magnetic separation on the reaction liquid containing the dsDNA-connected nano magnetic beads obtained in the step (4), removing a supernatant by using a pipette, and washing 3 times by 5mL of 50mM MES buffer containing 0.05% (v/v) Triton X-405, so as to obtain washed dsDNA-connected nano magnetic beads. The washed dsDNA-ligated nanobeads were stored in 10mL of 50mM MES buffer (pH = 7) containing 0.05% (v/v) Triton X-405 and 0.5% (v/v) Proclin 300.

Example 3

The reagents used to prepare dsDNA-ligated nanobeads in example 3 are shown in table 1, and the preparation method includes, but is not limited to, the following steps:

(1) 1mL of a carboxyl nanobead solution (the particle size of the carboxyl nanobead is 1 μm, the concentration is 100mg/mL, and the carboxyl content is 200nmol/mg, that is, each 1mL of the carboxyl nanobead solution contains 100mg of magnetic beads and each mg of the carboxyl nanobead contains 200nmol of carboxyl), treated in a magnetic separator for 3 minutes, removed the supernatant by a pipettor, and washed 3 times with 10mL of 100mM MES buffer (pH 6.5).

(2) Adding 2mL of 50mM MES buffer solution (pH 6.5) into the carboxyl nano-magnetic beads washed in the step (1), then adding 1mL of EDC solution (the concentration of EDC is 10 mg/mL) and 1mL of Sulfo-NHS solution (the concentration is 8 mg/mL), and rotating at 25 ℃ (40 r/min) for reaction for 1.5h to obtain activated carboxyl nano-magnetic beads.

(3) 6mL of a 5mg/mL dsDNA solution is prepared by using a 100mM phosphate buffer solution (pH is 8), and then the 6mL of the 5mg/mL dsDNA solution is bathed for 2h at 37 ℃ to obtain a preheated dsDNA solution.

(4) And (3) adding all the preheated dsDNA in the step (3) into the activated carboxyl magnetic beads in the step (2), controlling the pH of the mixed solution to be 7.0, adding 40 mu L of a DMSO solution of DMAP (the concentration of DMAP is 10 mg/mL), and rotating at 37 ℃ (60 revolutions per minute) for reacting for 24 hours to obtain a reaction solution containing the dsDNA-connected nano magnetic beads.

(5) And (4) performing magnetic separation on the reaction liquid containing the dsDNA-linked nano magnetic beads obtained in the step (4), and removing a supernatant by using a pipette. Then, the cells were washed 3 times with 5mL of 50mM PBS buffer containing 0.05% (v/v) Triton X-405. And obtaining the washed nano magnetic beads connected with the dsDNA. The washed dsDNA-ligated nanobeads were stored in 10mL of 50mM PBS buffer (pH = 7) containing 0.05% (v/v) Triton X-100 and 0.5% (v/v) Proclin 300.

Example 4

The reagents used to prepare dsDNA-ligated nanobeads in example 4 are shown in table 1, and the preparation method includes, but is not limited to, the following steps:

(1) 1mL of a solution of carboxyl nanobeads (the particle size of each carboxyl nanobead is 2 μm, the concentration is 100mg/mL, and the carboxyl content is 300nmol/mg, that is, each 1mL of the solution of carboxyl nanobeads contains 100mg of magnetic beads, and each mg of the solution of carboxyl nanobeads contains 300nmol of carboxyl groups) was treated in a magnetic separator for 3 minutes, the supernatant was removed by a pipette, and the solution was washed 3 times with 1mL of 100mM MES buffer (pH 6.5).

(2) Adding 2mL of 50mM MES buffer solution (pH 6.5) into the carboxyl nano magnetic beads washed in the step (1), then adding 2mL of EDC solution (the concentration of EDC is 10 mg/mL) and 2mL of NHS solution (the concentration of NHS is 8 mg/mL), and rotating at 25 ℃ (40 r/min) for reaction for 1.5h to obtain activated carboxyl nano magnetic beads.

(3) A dsDNA solution of 8mL of 5mg/mL was prepared using 100mM phosphate buffer (pH 7.8), and then the dsDNA solution of 8mL of 5mg/mL was subjected to a water bath at 50 ℃ for 1 hour to obtain a preheated dsDNA solution.

(4) And (3) adding all the preheated dsDNA solution in the step (3) into the activated carboxyl magnetic beads in the step (2), controlling the pH of the mixed solution to be 7.0, adding 20 mu L of a DMSO solution of DMAP (the concentration of DMAP is 10 mg/mL), and rotating (60 r/min) at 50 ℃ for reaction for 12h to obtain a reaction solution containing the dsDNA-connected nano magnetic beads.

(5) And (4) performing magnetic separation on the reaction liquid containing the dsDNA-linked nano magnetic beads obtained in the step (4), and removing a supernatant by using a pipette. Then, the cells were washed 3 times with 5mL of 50mM PBS buffer containing 0.1% (v/v) polyethylene glycol 400. And obtaining the cleaned nano magnetic beads connected with the dsDNA. The washed dsDNA-ligated nanobeads were stored in 10mL of 50mM PBS buffer (pH = 7.2) containing 0.05% (v/v) polyethylene glycol 400 and 0.2% (v/v) Proclin 950.

Example 5

The reagents used to prepare dsDNA-ligated nanobeads in example 5 were substantially the same as those used in example 2, except that the temperature and time of the water bath of the dsDNA solution and the reaction temperature of the dsDNA solution and the activated carboxyl nanobeads were different, and the reagents used in example 5 are specifically shown in table 1. The specific preparation of example 5 is substantially the same as that of example 2, except that the parameters indicated above have been adjusted accordingly.

Comparative example 1

The reagent used in the preparation of the dsDNA-attached magnetic fine particles in comparative example 1 was substantially the same as in example 1, except that no catalyst was used in comparative example 1, the reagent used in comparative example 1 was specifically shown in table 1, and the specific preparation method of comparative example 1 was substantially the same as in example 1, except that the step of adding the catalyst was omitted.

Comparative example 2

The reagents used in the preparation of dsDNA-attached magnetic microparticles in comparative example 2 were the same as in example 1, and are specifically shown in table 1. The dsDNA-attached magnetic fine particles of comparative example 2 were prepared in substantially the same manner as in example 1, except that the step of heat-treating the dsDNA solution was omitted in comparative example 2, and after preparing dsDNA into the dsDNA solution at room temperature (25 ℃), the mixing reaction of the dsDNA solution, 4-dimethylaminopyridine and activated carboxyl magnetic fine particles was directly performed at a mixing reaction temperature of 25 ℃.

Comparative example 3

The reagents used to prepare dsDNA-attached magnetic particles in comparative example 3 are the same as in example 2, as shown in table 1. Comparative example 3 the dsDNA-ligated magnetic particles were prepared in substantially the same manner as in example 2, except that in comparative example 3, the temperature of the water bath in the step of heat-treating the dsDNA solution was 20 ℃, the time of the water bath was 1.5 hours, and in the step of reacting the dsDNA solution with the carboxyl-based magnetic particles after activation, the reaction temperature was 20 ℃.

Comparative example 4

The reagents used in the preparation of dsDNA-attached magnetic microparticles in comparative example 4 were the same as in example 2, and are specifically shown in table 1. The dsDNA-attached magnetic fine particles of comparative example 4 were prepared in substantially the same manner as in example 2, except that in comparative example 4, the temperature of the water bath in the step of heat-treating the dsDNA solution was 65 ℃ and the water bath time was 1.5 hours, and in the step of reacting the dsDNA solution with the activated carboxyl magnetic fine particles, the reaction temperature was 65 ℃ and the reaction time was 18 hours.

Comparative example 5

The reagents used to prepare dsDNA-attached magnetic particles in comparative example 5 are the same as in example 2, as shown in table 1. The dsDNA-attached magnetic fine particles of comparative example 5 were prepared in substantially the same manner as in example 2, except that in comparative example 5, the reaction of the dsDNA solution with the carboxyl-based magnetic fine particles after activation was performed under the action of pyridine.

Test one

The dsDNA-connected nano magnetic beads obtained in each embodiment and each proportion are used for the chemiluminescence immunoassay detection of dsDNA antibodies, the test is completed in an iFlash3000 type chemiluminescence immunoassay analyzer, and the analyzer runs automatically according to a set program. Specific tests of the examples and comparative examples include, but are not limited to, the following steps:

(1) 1pmol of murine anti-human IgG acridine label was added to 50. Mu.L of a sample containing 300IU/mL dsDNA antibody and incubated at 37 ℃ for 10min, then 50ug of dsDNA-linked nanobeads of example 1 was added and incubated at 37 ℃ for 10min, followed by magnetic separation, washing 3 times, and finally 100. Mu.L of HNO was added ₃ -H ₂ O ₂ The luminescence value was measured for the solution and 100. Mu.L of sodium hydroxide solution. The measurements were performed in 3 replicates and averaged, the results are shown in table 1.

(2) The dsDNA-ligated nanobeads obtained in examples 2 to 5 and comparative examples 1 to 5 were subjected to the same procedure as in step 1, and the luminescence values corresponding to the dsDNA-ligated nanobeads obtained in examples 2 to 5 and comparative examples 1 to 5 were shown in table 1.

TABLE 1

As can be seen from Table 1, examples 1 to 5 all obtained higher luminescence values, indicating that the amount of dsDNA attached to the beads was large and the efficiency of coating the magnetic beads with dsDNA was high.

Compared with the example 1, the comparative example 1 has the advantage that only DMAP is not added when the nano magnetic beads connected with the dsDNA are prepared, the detected luminescence value is greatly different from that of the example 1, which shows that the coating efficiency of the dsDNA is lower when DMAP is not added, and the comparative example 5 shows that the reaction effect of pyridine on the activated carboxyl magnetic beads and the preheated dsDNA is not obvious, so that the coating efficiency of the dsDNA cannot be effectively improved.

In comparison with example 1, comparative example 2 did not perform the heating treatment and the esterification temperature did not increase when preparing the dsDNA-attached nanobead, however, the luminescence value of comparative example 2 was much different from that of example 1, and compared with comparative example 1 and example 1, it shows that the heating treatment and esterification temperature had a great influence on the efficiency of attaching dsDNA to the nanobead.

As can be seen from examples 1 to 5 and comparative examples 3 and 4, the coating efficiency of dsDNA at a bath temperature of 20 ℃ or 65 ℃ of the dsDNA solution is significantly lower than that at a bath temperature of 25 ℃ to 60 ℃.

As can be seen from example 2 and comparative example 5, 4-dimethylaminopyridine significantly improved dsDNA coating efficiency.

Test two

The dsDNA-linked nanobead obtained in example 1 was used for chemiluminescent immunoassay detection of dsDNA antibodies of different concentrations, and the test was performed in an iFlash3000 type chemiluminescent immunoassay analyzer, which was operated fully automatically according to a set program. Specifically, the method comprises the following steps:

dsDNA antibody samples with different concentrations are prepared, and the numbers are 1-6 respectively. Then, 1pmol of acridine label of mouse anti-human IgG was added to each sample, and the mixture was incubated at 37 ℃ for 10min. Then 50ug of dsDNA nanobeads obtained in example 1 were added and incubated at 37 ℃ for 10min, followed by magnetic separation, washing 3 times, and finally 100. Mu.L of HNO was added ₃ -H ₂ O ₂ The luminescence value was measured using the solution and 100. Mu.L of sodium hydroxide solution. 3 measurements were carried out in parallelThe results are shown in Table 2.

TABLE 2

Number of	dsDNA antibody concentration (IU/mL)	Mean luminous value (RLU)
			1	8	6808
2	16	11883
			3	48	22234
4	96	37267
			5	144	59845
6	288	103690

The average luminous value of each sample and the correspondingConcentration is fitted straight to give curve y =345.77x +5710.8 ² =0.9959, results are shown in fig. 2. In FIG. 2, the horizontal axis represents dsDNA antibody concentration (IU/mL) and the vertical axis Represents Luminescence (RLU).

As shown in Table 2, the dsDNA-ligated nanobeads prepared in example 1 were linear when used in chemiluminescent immunoassay detection of dsDNA antibodies.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method of preparing magnetic particles having dsDNA attached thereto, comprising the steps of:

activating carboxyl groups of the carboxyl magnetic particles to prepare activated carboxyl magnetic particles;

preheating a solution containing dsDNA for 0.5 to 3 hours at the temperature of 25 to 60 ℃ to prepare preheated dsDNA; and

under the condition of 25-60 ℃, 4-dimethylamino pyridine, the activated carboxyl magnetic particles and the preheated dsDNA are mixed and reacted to prepare magnetic particles connected with the dsDNA; and activating the carboxyl of the carboxyl magnetic particles by using carbodiimide and hydroxysuccinimide.

2. The method of claim 1, wherein the preheating treatment of the dsDNA containing solution is performed at a temperature of 35 ℃ to 50 ℃.

3. The method of claim 1, wherein the dsDNA-containing solution is subjected to heat treatment for 1 to 2h.

4. The method as claimed in claim 1, wherein in the step of mixing 4-dimethylaminopyridine, the activated carboxyl magnetic particles and the preheated dsDNA, the temperature of the mixing reaction is 35 ℃ to 50 ℃.

5. The production method according to claim 1, wherein the molar ratio of 4-dimethylaminopyridine to carboxyl groups in the activated carboxyl magnetic particles is (0.05 to 0.3): 1.

6. the method according to claim 1, wherein in the step of mixing and reacting the 4-dimethylaminopyridine, the activated carboxyl magnetic particles and the preheated dsDNA, the pH of a mixture of the activated carboxyl magnetic particles and the preheated dsDNA is 6 to 7.5.

7. The method according to claim 1, wherein the carboxyl magnetic fine particles are carboxyl nanobeads, and the particle size of the carboxyl magnetic fine particles is 0.5 to 3 μm.

8. The method as claimed in claim 1, wherein in the step of mixing and reacting 4-dimethylaminopyridine, the activated carboxyl-based magnetic particles and the preheated dsDNA, the mass of the preheated dsDNA is 0.05-2 times the mass of the activated carboxyl-based magnetic particles.

9. The method for preparing the peptide of 1~8, wherein the dsDNA solution is prepared by dissolving dsDNA in a buffer, the pH of the dsDNA solution is 7 to 8.5, and the buffer does not contain amino, hydroxyl or thiol.

10. The method according to claim 9, wherein the buffer is at least one selected from the group consisting of a phosphate buffer, a carbonate buffer, and a borate buffer.