Synthesis method and application of multifunctional magnetic beads

Abstract

The invention provides a synthetic method of magnetic beads. In a magnetic bead synthesis path provided by the invention, the carboxyl density is improved in a branching amplification manner, and then post-crosslinking is performed by utilizing a Friedel Crafts reaction to ensure that the specific surface area of the microspheres is increased; meanwhile, a branching amplification mode can effectively avoid dependence on the chlorine content in a polymeric monomer, and the magnetic microspheres with high specific surface area and high carboxyl density are finally obtained. The prepared multifunctional magnetic beads are suitable for extraction of two items, namely steroid hormones and catecholamine metabolites, the steroid hormones can be extracted firstly, then the catecholamine metabolites can be extracted, and the steroid hormones and the catecholamine metabolites can also be extracted at the same time. The kit prepared by adopting the magnetic beads provided by the invention is simple in composition, the pretreatment is more flexible, two items can be taken into account, the eluent can meet the sample introduction requirements of steroid hormone reversed-phase chromatography and catecholamine metabolite normal-phase chromatography, and the solvent effect is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of nano magnetic beads for the biological industry, and relates to a method for synthesizing magnetic beads and its application, in particular to a method for synthesizing multifunctional magnetic beads and its application. Background Art

[0002] Due to its unique superparamagnetism, magnetic beads can be used as an extraction, separation and purification carrier, which can bind and adsorb proteins and small molecule substances in biological samples such as blood, serum, urine, etc. Further, through the purification, elution, dispersion of the target substance or the removal of impurities from the target substance, the analysis and detection of the target component can be realized.

[0003] At present, magnetic bead extraction is not only applied to nucleic acid extraction, but also used for the extraction of the pretreatment of detection items on the mass spectrometry platform. For the detection of catecholamines and their metabolites, the currently common type of extraction magnetic beads is the weakly cation-exchange WCX magnetic beads, and for steroid hormone projects, they are the hydrophilic-lipophilic HLB magnetic beads. However, there is currently no magnetic bead that can be applied to the extraction of both projects. For example, in the publication number CN 201880039549, a method is described in which magnetic iron oxide is used as the core, and large-sized particles are obtained through suspension polymerization, and then post-modification is carried out on the surface of the hypercrosslinked particles obtained through the Friedel Crafts reaction to obtain functional groups. However, due to the limited post-modification methods and all being carried out based on the reaction with the residual chlorine after the hypercrosslinking reaction, the density of the obtained functional groups is insufficient, which will affect the adsorption of the target substance. Regarding the problem of functional group density, borrowing the synthesis method of dendritic polymers and using the convergent method or the divergent method for branching amplification is a common means. For example, in the publication number CN 201911278010, the functionalization of the surface of magnetic microspheres is branched and introduced by using the classical amide reaction mechanism, so that the density of the functional groups on the surface of the magnetic microspheres is greatly improved. However, the specific surface area of the microspheres obtained by this method is usually small, which may also affect the adsorption of the target substance. Therefore, it is still necessary to develop a series of useful magnetic particles, especially magnetic particles with a high specific surface area and a high functional group density that are beneficial to the liquid chromatography-mass spectrometry system.

[0004] At the same time, the current magnetic bead synthesis method cannot guarantee a high specific surface area and a high energy group density, nor is there a corresponding technical solution for magnetic beads and kit products that can quickly extract catecholamines and steroid hormone projects.

[0005] Therefore, how to find a more suitable method for preparing magnetic beads to solve the above problems existing in the prior art and be able to be used for quickly extracting catecholamines and steroid hormone projects has become one of the urgent problems to be solved by many R & D manufacturers and front-line researchers. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for synthesizing magnetic beads and its application, in particular a method for synthesizing multifunctional magnetic beads. The multifunctional magnetic beads prepared by the present invention can be used for quickly extracting catecholamines and steroid hormones, and the extraction sequence is relatively flexible, which improves the convenience of use, reduces the cost of the kit, and has a simple production process, mild conditions, good controllability, and is more suitable for the popularization and application of industrial production, and has good application prospects in the field of biomedicine.

[0007] The present invention provides a method for synthesizing magnetic beads, comprising the following steps:

[0008] 1) Mix the aqueous dispersion of magnetic nanoparticles with oleic acid, react and then separate to obtain magnetic cores coated with oleic acid;

[0009] 2) Mix the magnetic cores coated with oleic acid obtained in the above step, the first polymerization monomer, the second polymerization monomer, the oil-phase solvent and the initiator to obtain an oil-phase mixed solution;

[0010] 3) Add the oil-phase mixed solution obtained in the above step to the aqueous dispersion of polyvinyl alcohol for reaction to obtain magnetic polymer microspheres with a copolymer coating on the surface;

[0011] 4) React the magnetic polymer microspheres obtained in the above step with an alkali solution, and then mix with an oxidant for a second reaction to obtain carboxyl-modified magnetic polymer microspheres;

[0012] 5) Mix and react the carboxyl-modified magnetic polymer microspheres obtained in the above step, a diamine compound and a first solvent to obtain amino-modified microspheres, and then mix and react the amino-modified microspheres, an acrylate compound and the first solvent again to obtain multi-ester-based microspheres;

[0013] 6) React the multi-ester-based microspheres obtained in the above step in a solvent containing a Lewis base to obtain microspheres with amplified carboxyl density;

[0014] 7) Under the condition of a protective atmosphere and the action of a Lewis acid catalyst, mix and crosslink the microspheres with amplified carboxyl density obtained in the above step, a crosslinking agent and a second solvent to obtain magnetic beads.

[0015] Preferably, the magnetic nanoparticles include Fe 3 O 4 nanoparticles;

[0016] The particle size of the magnetic nanoparticles is 5-20 nm;

[0017] The pH value of the aqueous dispersion of the magnetic nanoparticles is 7-9;

[0018] The mixing reaction in step 1) specifically involves dropping oleic acid into an aqueous dispersion of magnetic nanoparticles for reaction;

[0019] The magnetic polymer microspheres specifically refer to magnetic polymer microspheres with a copolymer coating on the surface.

[0020] Preferably, the mass ratio of the oleic acid-coated magnetic core to the first polymerization monomer is (1 - 5):(3 - 6);

[0021] The first polymerization monomer includes divinylbenzene;

[0022] The second polymerization monomer includes vinylbenzyl chloride;

[0023] The volume ratio of the first polymerization monomer to the second polymerization monomer is 1:(1 - 3);

[0024] The initiator includes azobisisobutyronitrile;

[0025] The mass ratio of azobisisobutyronitrile to the first polymerization monomer is 1:(1 - 15).

[0026] Preferably, the oil-phase solvent includes one or more of 1,2-dichloroethane, toluene, xylene, and dichloromethane;

[0027] The content of polyvinyl alcohol in the polyvinyl alcohol aqueous dispersion is 1 wt% - 10 wt%;

[0028] The volume ratio of the oil-phase mixed solution to the polyvinyl alcohol aqueous dispersion is 1:(2 - 6);

[0029] The adding method includes dropping;

[0030] In step 3), the reaction temperature is 60 - 100 °C;

[0031] In step 3), the reaction time is 2 - 8 h;

[0032] The magnetic polymer microspheres include magnetic polydivinylbenzene-vinylbenzyl chloride copolymer microspheres.

[0033] Preferably, the alkali solution includes sodium hydroxide solution;

[0034] The temperature of the first reaction is 20 - 80 °C;

[0035] The time of the first reaction is 4 - 16 h;

[0036] The oxidant includes sodium hypochlorite;

[0037] The temperature of the second reaction is 40 - 80 °C;

[0038] The time of the second reaction is 1 to 4 h.

[0039] Preferably, step 4) can also be the following steps:

[0040] 4`) After carrying out a nucleophilic substitution reaction on the magnetic polymer microspheres, aromatic carboxylic acid monomers and an alkali solution obtained in the above steps, carboxyl-modified magnetic polymer microspheres are obtained;

[0041] The diamine compounds include one or more of ethylenediamine, propylenediamine and butylenediamine;

[0042] The mass-volume ratio of the magnetic polymer microspheres to the diamine compounds is 1 g:(1 - 4) mL;

[0043] The first solvent is selected from one or more of alcohol solvents, ether solvents, ketone solvents, nitrile solvents and pyridine;

[0044] The time of the mixing reaction is 12 to 48 h;

[0045] The acrylate compounds include one or more of methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, n-butyl acrylate, tert-butyl acrylate, isobutyl acrylate, n-pentyl acrylate, isopentyl acrylate and hexyl acrylate;

[0046] The mass-volume ratio of the amino-modified microspheres to the acrylate compounds is 1 g:(1 - 5) mL.

[0047] Preferably, the time of the re-mixing reaction is 12 to 48 h;

[0048] Step 5) is specifically carried out repeatedly;

[0049] The number of times is 3 to 5 times;

[0050] The Lewis bases include one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium oxide and calcium oxide;

[0051] In the solvent containing a Lewis base, the solvent is selected from one or more of alcohol solvents, ether solvents, ketone solvents, nitrile solvents and pyridine;

[0052] The mass ratio of the multi-ester-based microspheres to the Lewis base is (1 - 7):1;

[0053] The Lewis acid catalysts include one or more of ferric chloride, zinc chloride and aluminum trichloride.

[0054] Preferably, the mixing in step 7) is specifically as follows: first, the microspheres with amplified carboxyl density are mixed and swollen with a second solvent. After swelling, they are mixed again with a crosslinking agent and a Lewis acid catalyst and then subjected to a crosslinking reaction;

[0055] The swelling time is 0.5 - 2 h;

[0056] The mass ratio of the microspheres with amplified carboxyl density to the Lewis acid catalyst is (1 - 7):1;

[0057] The crosslinking agent includes dimethoxymethane;

[0058] The mass ratio of the microspheres with amplified carboxyl density to the crosslinking agent is (1 - 5):1;

[0059] The second solvent includes one or more of 1,2 - dichloroethane, toluene, N,N - dimethylformamide, dimethyl sulfoxide, and dichloromethane;

[0060] The crosslinking reaction time is 2 - 48 h.

[0061] Preferably, the magnetic beads are specifically surface - porous magnetic microspheres with a high specific surface area and a high carboxyl density;

[0062] The particle size of the magnetic beads is 1 - 100 μm;

[0063] The magnetic beads have superparamagnetism;

[0064] The specific surface area of the magnetic beads is ≥200 m 2 / g;

[0065] At least 80% of the pore diameters of the magnetic beads are ≤5 nm;

[0066] The carboxyl content on the surface of the magnetic beads is 0.3 - 2 mmol / g;

[0067] The saturation magnetization intensity of the magnetic beads is ≥4 emu / g.

[0068] The present invention also provides the application of the magnetic beads prepared by the synthesis method according to any one of the above - mentioned technical solutions in extracting and / or detecting steroid hormones and / or catecholamine metabolites.

[0069] The present invention provides a method for synthesizing magnetic beads, which includes the following steps: First, a water dispersion of magnetic nanoparticles is mixed with oleic acid for reaction and then separated to obtain magnetic cores coated with oleic acid; Then, the magnetic cores coated with oleic acid obtained in the above step, a first polymerization monomer, a second polymerization monomer, an oil-phase solvent and an initiator are mixed to obtain an oil-phase mixed solution; Next, the oil-phase mixed solution obtained in the above step is added to a polyvinyl alcohol water dispersion for reaction to obtain magnetic polymer microspheres with a copolymer-coated surface; Subsequently, the magnetic polymer microspheres obtained in the above step and an alkali solution are subjected to a first reaction, and then mixed with an oxidant for a second reaction to obtain carboxyl-modified magnetic polymer microspheres; Then, the carboxyl-modified magnetic polymer microspheres obtained in the above step, a diamine compound and a first solvent are mixed for reaction to obtain amino-modified microspheres, and then the amino-modified microspheres, an acrylate compound and a first solvent are mixed again for reaction to obtain multi-ester-based microspheres; Next, the multi-ester-based microspheres obtained in the above step are reacted in a solvent containing a Lewis base to obtain microspheres with amplified carboxyl density; Finally, under the conditions of a protective atmosphere and the action of a Lewis acid catalyst, the carboxyl density-amplified microspheres obtained in the above step, a crosslinking agent and a second solvent are mixed for crosslinking reaction to obtain magnetic beads. Compared with the prior art, the present invention adopts specific preparation steps to obtain magnetic microspheres with a high specific surface area and a high carboxyl density. In the magnetic bead synthesis route provided by the present invention, the carboxyl density is first increased by means of branched amplification, and then Friedel Crafts reaction is used for post-crosslinking to ensure an increase in the specific surface area of the microspheres; at the same time, the branched amplification method can effectively avoid dependence on the chlorine content in the polymerization monomer.

[0070] The multifunctional magnetic beads prepared by the method for preparing magnetic microspheres with a high specific surface area and a high carboxyl density provided by the present invention are applicable to the extraction of two items, namely steroid hormones and catecholamine metabolites. Steroid hormones can be extracted first, and then catecholamine metabolites (the order cannot be reversed here), or steroid hormones and catecholamine metabolites can be extracted simultaneously.

[0071] The kit prepared by using the multifunctional magnetic beads prepared by the present invention has a simple composition and more flexible pretreatment. It can take both items into account, and the eluent can meet the injection requirements of reverse-phase chromatography for steroid hormones and normal-phase chromatography for catecholamine metabolites, avoiding solvent effects. Description of the Drawings

[0072] Figure 1 It is a morphology diagram of the magnetic microspheres with a high specific surface area and a high carboxyl density prepared by the present invention;

[0073] Figure 2 It is a saturation magnetization intensity data diagram of the magnetic microspheres with a high specific surface area and a high carboxyl density prepared by the present invention;

[0074] Figure 3 Characteristic diagram of magnetic microspheres with high specific surface area and high carboxyl density prepared according to the present invention;

[0075] Figure 4 Magnetic response time curve diagram of magnetic microspheres with high specific surface area and high carboxyl density prepared according to the present invention;

[0076] Figure 5 Potentiometric titration curve diagram of magnetic microspheres with high specific surface area and high carboxyl density prepared according to the present invention;

[0077] Figure 6 Chromatogram of 17-OHP and 17-OHP-13C3 in Application Scheme 1 of the present invention;

[0078] Figure 7 Chromatogram of DHT and DHT-D3 in Application Scheme 1 of the present invention;

[0079] Figure 8 Chromatogram of A4 and A4-13C3 in Application Scheme 1 of the present invention;

[0080] Figure 9 Chromatogram of T and T-13C3 in Application Scheme 1 of the present invention;

[0081] Figure 10 Chromatogram of DHEAS and DHEAS-D6 in Application Scheme 1 of the present invention;

[0082] Figure 11 Chromatogram of MN and MN-d3 in Application Scheme 2 of the present invention;

[0083] Figure 12 Chromatogram of NMN and NMN-d3 in Application Scheme 2 of the present invention;

[0084] Figure 13 Chromatogram of 3-MT and 3-MT-d4 in Application Scheme 2 of the present invention. Detailed implementation manners

[0085] To further understand the present invention, the preferred implementation schemes of the present invention are described below in conjunction with embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0086] For all raw materials of the present invention, there is no particular limitation on their sources, and they can be purchased on the market or prepared according to conventional methods well-known to those skilled in the art.

[0087] For all raw materials of the present invention, there is no particular limitation on their purity. The present invention preferably adopts analytical purity or the conventional purity requirements for preparing magnetic beads in the biological field.

[0088] All raw materials of the present invention, their grades and abbreviations belong to the conventional grades and abbreviations in the art, and each grade and abbreviation is clear and definite in the field of its related uses. Those skilled in the art can purchase them from the market or prepare them by conventional methods according to the grade, abbreviation and corresponding uses.

[0089] The present invention provides a method for synthesizing magnetic beads, comprising the following steps:

[0090] 1) Mix the aqueous dispersion of magnetic nanoparticles with oleic acid, react and then separate to obtain magnetic cores coated with oleic acid;

[0091] 2) Mix the magnetic cores coated with oleic acid obtained in the above step, the first polymerization monomer, the second polymerization monomer, the oil-phase solvent and the initiator to obtain an oil-phase mixed solution;

[0092] 3) Add the oil-phase mixed solution obtained in the above step to the aqueous dispersion of polyvinyl alcohol for reaction to obtain magnetic polymer microspheres with a copolymer coating on the surface;

[0093] 4) React the magnetic polymer microspheres obtained in the above step with an alkali solution, and then mix with an oxidant for a second reaction to obtain carboxyl-modified magnetic polymer microspheres;

[0094] 5) Mix and react the carboxyl-modified magnetic polymer microspheres obtained in the above step, the diamine compound and the first solvent to obtain amino-modified microspheres, and then mix and react the amino-modified microspheres, the acrylate compound and the first solvent again to obtain multi-ester-based microspheres;

[0095] 6) React the multi-ester-based microspheres obtained in the above step in a solvent containing a Lewis base to obtain microspheres with amplified carboxyl density;

[0096] 7) Under the condition of a protective atmosphere and the action of a Lewis acid catalyst, mix the microspheres with amplified carboxyl density obtained in the above step, the cross-linking agent and the second solvent for cross-linking reaction to obtain magnetic beads.

[0097] The present invention first mixes the aqueous dispersion of magnetic nanoparticles with oleic acid, reacts and then separates to obtain magnetic cores coated with oleic acid.

[0098] In the present invention, the magnetic nanoparticles preferably include Fe 3 O 4 nanoparticles.

[0099] In the present invention, the particle size of the magnetic nanoparticles is preferably 5-20 nm, more preferably 7-18 nm, and even more preferably 9-16 nm.

[0100] In the present invention, the pH value of the aqueous dispersion of the magnetic nanoparticles is preferably 7 to 9, more preferably 7.4 to 8.6, and even more preferably 7.8 to 8.2.

[0101] In the present invention, the mixing reaction in step 1) is specifically preferably carried out by dropping oleic acid into the aqueous dispersion of magnetic nanoparticles for reaction.

[0102] In the present invention, the oleic acid-coated magnetic core, the first polymerization monomer, the second polymerization monomer, the oil-phase solvent and the initiator obtained in the above steps are then mixed to obtain an oil-phase mixed solution.

[0103] In the present invention, the mass ratio of the oleic acid-coated magnetic core to the first polymerization monomer is preferably (1 to 5):(3 to 6), more preferably (1.5 to 4.5):(3.5 to 5.5), and even more preferably (2 to 4):(4 to 5).

[0104] In the present invention, the first polymerization monomer preferably includes divinylbenzene.

[0105] In the present invention, the second polymerization monomer preferably includes vinylbenzyl chloride.

[0106] In the present invention, the volume ratio of the first polymerization monomer to the second polymerization monomer is preferably 1:(1 to 3), more preferably 1:(1.4 to 2.6), and even more preferably 1:(1.8 to 2.2).

[0107] In the present invention, the initiator preferably includes azobisisobutyronitrile.

[0108] In the present invention, the mass ratio of azobisisobutyronitrile to the first polymerization monomer is preferably 1:(1 to 15), more preferably 1:(4 to 12), and even more preferably 1:(7 to 9).

[0109] In the present invention, the oil-phase solvent preferably includes one or more of 1,2-dichloroethane, toluene, xylene and dichloromethane, and more preferably 1,2-dichloroethane, toluene, xylene or dichloromethane.

[0110] In the present invention, the oil-phase mixed solution obtained in the above steps is then added to the polyvinyl alcohol aqueous dispersion for reaction to obtain magnetic polymer microspheres with a copolymer-coated surface.

[0111] In the present invention, the magnetic polymer microspheres are specifically preferably magnetic polymer microspheres with a copolymer-coated surface.

[0112] In the present invention, the content of polyvinyl alcohol in the polyvinyl alcohol aqueous dispersion is preferably 1 wt% to 10 wt%, more preferably 3 wt% to 8 wt%, and even more preferably 5 wt% to 6 wt%.

[0113] In the present invention, the volume ratio of the oil-phase mixed solution to the polyvinyl alcohol aqueous dispersion is preferably 1:(2 - 6), more preferably 1:(2.5 - 5.5), and even more preferably 1:(3 - 5).

[0114] In the present invention, the addition method preferably includes dropping addition.

[0115] In the present invention, in step 3), the reaction temperature is preferably 60 - 100 °C, more preferably 65 - 95 °C, even more preferably 70 - 90 °C, and even more preferably 75 - 85 °C, and specifically can be 80 °C.

[0116] In the present invention, in step 3), the reaction time is preferably 2 - 8 h, more preferably 3 - 7 h, even more preferably 4 - 6 h, and specifically can be 4 h.

[0117] In the present invention, the magnetic polymer microspheres preferably include magnetic polydivinylbenzene - vinylbenzyl chloride copolymer microspheres.

[0118] Subsequently, in the present invention, the magnetic polymer microspheres obtained in the above steps and an alkali solution are subjected to a first reaction, and then mixed with an oxidant for a second reaction to obtain carboxyl - modified magnetic polymer microspheres.

[0119] In the present invention, the alkali solution preferably includes a sodium hydroxide solution.

[0120] In the present invention, the temperature of the first reaction is preferably 20 - 80 °C, more preferably 30 - 70 °C, even more preferably 40 - 60 °C, and specifically can be 60 °C.

[0121] In the present invention, the time of the first reaction is preferably 4 - 16 h, more preferably 6 - 14 h, even more preferably 6 - 14 h, and even more preferably 8 - 12 h, and specifically can be 8 h.

[0122] In the present invention, the oxidant preferably includes sodium hypochlorite.

[0123] In the present invention, the temperature of the second reaction is preferably 40 - 80 °C, more preferably 45 - 75 °C, even more preferably 50 - 70 °C, and even more preferably 55 - 65 °C, and specifically can be 60 °C.

[0124] In the present invention, the time of the second reaction is preferably 1 - 4 h, more preferably 1.5 - 3 h, even more preferably 1.5 - 2 h, and specifically can be 1.5 h.

[0125] In the present invention, step 4) can also preferably be the following steps:

[0126] 4`) After subjecting the magnetic polymer microspheres, aromatic carboxylic acid monomers, and alkaline solution obtained in the above steps to a nucleophilic substitution reaction, carboxyl-modified magnetic polymer microspheres are obtained.

[0127] In the present invention, the carboxyl-modified magnetic polymer microspheres, diamine compounds, and a first solvent obtained in the above steps are then mixed and reacted to obtain amino-modified microspheres. Then, the amino-modified microspheres, acrylate compounds, and the first solvent are mixed and reacted again to obtain multi-ester-based microspheres.

[0128] In the present invention, the diamine compounds preferably include one or more of ethylenediamine, propylenediamine, and butylenediamine, and more preferably ethylenediamine, propylenediamine, or butylenediamine.

[0129] In the present invention, the mass-to-volume ratio of the magnetic polymer microspheres to the diamine compounds is preferably 1 g : (1 - 4) mL, more preferably 1 g : (1.5 - 3.5) mL, and even more preferably 1 g : (2 - 3) mL.

[0130] In the present invention, the first solvent is preferably selected from one or more of alcohol solvents, ether solvents, ketone solvents, nitrile solvents, and pyridine, and more preferably an alcohol solvent, an ether solvent, a ketone solvent, a nitrile solvent, or pyridine, and specifically can be one or more of methanol, ethanol, methyl ether, ethyl ether, etc.

[0131] In the present invention, the reaction time for the mixing reaction is preferably 12 - 48 h, more preferably 17 - 43 h, even more preferably 22 - 38 h, and even more preferably 27 - 33 h, and specifically can be 24 h.

[0132] In the present invention, the acrylate compounds preferably include one or more of methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, n-butyl acrylate, tert-butyl acrylate, isobutyl acrylate, n-pentyl acrylate, isopentyl acrylate, and hexyl acrylate, and more preferably methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, n-butyl acrylate, tert-butyl acrylate, isobutyl acrylate, n-pentyl acrylate, isopentyl acrylate, or hexyl acrylate.

[0133] In the present invention, the mass-to-volume ratio of the amino-modified microspheres to the acrylate compounds is preferably 1 g : (1 - 5) mL, more preferably 1.5 - 3 h, and even more preferably 1.5 - 2 h.

[0134] In the present invention, the reaction time for the re-mixing reaction is preferably 12 - 48 h, more preferably 17 - 43 h, even more preferably 22 - 38 h, and even more preferably 27 - 33 h, and specifically can be 24 h.

[0135] In the present invention, step 5) is specifically preferably carried out repeatedly multiple times.

[0136] In the present invention, the number of times of the multiple times is preferably 3 to 5 times, more preferably 3 times, 4 times or 5 times.

[0137] In the present invention, the multi-ester group microspheres obtained in the above steps are reacted in a solvent containing a Lewis base to obtain microspheres with amplified carboxyl density.

[0138] In the present invention, the Lewis base preferably includes one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium oxide and calcium oxide, and more preferably lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium oxide or calcium oxide.

[0139] In the present invention, in the solvent containing a Lewis base, the solvent is preferably selected from one or more of alcohol solvents, ether solvents, ketone solvents, nitrile solvents and pyridine, more preferably alcohol solvents, ether solvents, ketone solvents, nitrile solvents or pyridine, and specifically can be one or more of methanol, ethanol, methyl ether, ethyl ether, etc.

[0140] In the present invention, the mass ratio of the multi-ester group microspheres to the Lewis base is preferably (1 to 7):1, more preferably (2 to 6):1, and more preferably (3 to 5):1.

[0141] In the present invention, the Lewis acid catalyst preferably includes one or more of ferric chloride, zinc chloride and aluminum trichloride, and more preferably ferric chloride, zinc chloride or aluminum trichloride.

[0142] Finally, in the present invention, under the condition of a protective atmosphere and the action of a Lewis acid catalyst, the microspheres with amplified carboxyl density, a crosslinking agent and a second solvent obtained in the above steps are mixed to carry out a crosslinking reaction to obtain magnetic beads.

[0143] In the present invention, the mixing in step 7) is specifically preferably that the microspheres with amplified carboxyl density are first mixed and swollen with the second solvent, and after swelling, they are mixed again with the crosslinking agent and the Lewis acid catalyst and then subjected to a crosslinking reaction.

[0144] In the present invention, the swelling time is preferably 0.5 to 2 h, more preferably 0.5 to 1.5 h, more preferably 0.5 to 1 h, and specifically can be 0.5 h.

[0145] In the present invention, the mass ratio of the microspheres with amplified carboxyl density to the Lewis acid catalyst is preferably (1 to 7):1, more preferably (2 to 6):1, and more preferably (3 to 5):1.

[0146] In the present invention, the crosslinking agent preferably includes dimethoxymethane.

[0147] In the present invention, the mass ratio of the carboxyl group density-amplified microspheres to the crosslinking agent is preferably (1-5):1, more preferably (1.5-4.5):1, still more preferably (2-4):1, and even more preferably (2.5-3.5):1.

[0148] In the present invention, the second solvent preferably includes one or more of 1,2-dichloroethane, toluene, N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane, and more preferably 1,2-dichloroethane, toluene, N,N-dimethylformamide, dimethyl sulfoxide, or dichloromethane.

[0149] In the present invention, the time of the crosslinking reaction is preferably 2-48 h, more preferably 4-28 h, still more preferably 4-8 h. Specifically, it can be 4 h.

[0150] In the present invention, the magnetic beads are specifically preferably surface-porous magnetic microspheres with a high specific surface area and a high carboxyl group density.

[0151] In the present invention, the particle size of the magnetic beads is preferably 1-100 μm, more preferably 5-80 μm, still more preferably 10-60 μm, and even more preferably 20-50 μm.

[0152] In the present invention, the magnetic beads preferably have superparamagnetism.

[0153] In the present invention, the specific surface area of the magnetic beads is preferably ≥200 m 2 / g, more preferably ≥300 m 2 / g, still more preferably ≥600 m 2 / g, and specifically it can be 700 m 2 / g.

[0154] In the present invention, at least 80% of the pore diameter of the magnetic beads is preferably ≤5 nm, more preferably ≤4.5 nm, still more preferably ≤4 nm, and specifically it can be 4 nm.

[0155] In the present invention, the carboxyl group content on the surface of the magnetic beads is preferably 0.3-2 mmol / g, more preferably 0.61-1.5 mmol / g, still more preferably 0.61-1.0 mmol / g, and specifically it can be 0.61 mmol / g.

[0156] In the present invention, the saturation magnetization intensity of the magnetic beads is preferably ≥4 emu / g, more preferably ≥5 emu / g, still more preferably ≥6 emu / g, and specifically it can be 6.1 emu / g.

[0157] The present invention provides the application of the magnetic beads prepared by the synthesis method according to any one of the above technical solutions in extracting and / or detecting steroid hormones and / or catecholamine metabolites.

[0158] The present invention also provides a kit, which comprises magnetic beads prepared by the synthesis method described in any one of the above technical solutions.

[0159] In the present invention, the kit preferably comprises a kit for extracting and / or detecting steroid hormones and / or catecholamine metabolites.

[0160] To complete and refine the overall technical solution of the present invention and better improve the effect of the multifunctional magnetic beads in the project of extracting or detecting steroid hormones and / or catecholamine metabolites, the synthesis method and application of the above multifunctional magnetic beads may specifically include the following contents:

[0161] The present invention provides a magnetic bead synthesis path for the extraction of steroid hormones and catecholamine metabolites and its kit application scheme.

[0162] The synthesis path includes the following steps:

[0163] (1) Preparation of magnetic polymer particles: Prepare magnetic cores by methods well-known in the industry, and then perform suspension polymerization with at least two different monomers to obtain magnetic polymer particles.

[0164] (2) Carboxyl modification: The obtained magnetic polymer particles undergo a nucleophilic substitution reaction under alkaline conditions to obtain hydroxyl-modified polymers, and then are further oxidized to obtain carboxyl-modified magnetic polymer microspheres. Or the magnetic polymer microspheres are subjected to a nucleophilic substitution reaction with aromatic carboxylic acid monomers under alkaline conditions to obtain carboxyl-modified magnetic polymer microspheres.

[0165] (3) Carboxyl density amplification:

[0166] 3a: Suspend the carboxyl-modified magnetic polymer microspheres in a first solvent, and add a diamine compound to react to obtain amino-modified microspheres

[0167] 3b: Suspend the product obtained in 3a in the first solvent again, and add an acrylate compound to react to obtain polyester-based microspheres;

[0168] Repeat the above 3a and 3b, cycle 3 to 5 times, and then place the obtained magnetic microspheres in a first solvent containing a Lewis base for stirring reaction at room temperature, and then perform magnetic separation and washing to obtain magnetic polymer microspheres with an amplified surface carboxyl density.

[0169] (4) Increase in specific surface area: Crosslink the magnetic microspheres obtained in (3) under the catalysis of a Lewis acid to obtain magnetic polymer microspheres with a high specific surface area and a high carboxyl density.

[0170] The above content of the present invention provides a method for synthesizing a multifunctional magnetic bead and its application. The present invention adopts specific preparation steps to obtain magnetic microspheres with a high specific surface area and a high carboxyl density. In the magnetic bead synthesis path provided by the present invention, the carboxyl density is first increased by a method of branched amplification, and then post-crosslinking is carried out by using the Friedel Crafts reaction to ensure an increase in the specific surface area of the microspheres; at the same time, the method of branched amplification can effectively avoid dependence on the chlorine content in the polymerization monomer.

[0171] The multifunctional magnetic beads prepared by the method for preparing magnetic microspheres with a high specific surface area and a high carboxyl density provided by the present invention are applicable to the extraction of two items, namely steroid hormones and catecholamine metabolites. Steroid hormones can be extracted first, and then catecholamine metabolites can be extracted (the order here cannot be reversed), or steroid hormones and catecholamine metabolites can be extracted simultaneously.

[0172] The kit prepared by using the multifunctional magnetic beads prepared by the present invention has a simple composition and more flexible pretreatment, can take into account the two items, and the eluent can meet the injection requirements of reverse-phase chromatography for steroid hormones and normal-phase chromatography for catecholamine metabolites, avoiding the occurrence of solvent effects.

[0173] In order to further illustrate the present invention, the following takes examples to describe in detail a method for synthesizing a magnetic bead and its application provided by the present invention. However, it should be understood that these examples are implemented on the premise of the technical solution of the present invention, and give detailed implementation manners and specific operation processes, only to further illustrate the features and advantages of the present invention, rather than a limitation on the claims of the present invention. The protection scope of the present invention is not limited to the following examples.

[0174] Example 1

[0175] The specific magnetic bead synthesis scheme is as follows:

[0176] Synthesis of oleic acid-coated Fe 3 O 4 Magnetic core: Under nitrogen protection, at 55 °C, ammonia water is added to an aqueous solution containing ferrous chloride and ferric chloride in a molar ratio of 1:2. After reacting for 30 minutes, the obtained Fe 3 O 4 nanoparticles are washed 3 times with pure water, and then redispersed in water, and the pH is adjusted to 7-9. Subsequently, oleic acid is added dropwise, and after 45 minutes, it is separated with a magnet and washed alternately with pure water and absolute ethanol to obtain oleic acid-coated Fe 3 O 4 magnetic core.

[0177] 1. Preparation of magnetic polymer microspheres (PDVB-VBC):

[0178] Mix 2.5 g of magnetic nuclei with 2 mL of divinylbenzene, 2 mL of vinylbenzyl chloride, and 20 mL of 1,2-dichloroethane, ultrasonicate for 50 minutes, and then add 0.2 g of azobisisobutyronitrile to obtain Solution A; Disperse 1.8 g of polyvinyl alcohol in 80 mL of water and dissolve it by stirring at 80 °C to obtain Solution B. Under the condition of 80 °C, drop Solution A into Solution B and stir for reaction for 4 h. After the reaction is completed, separate the obtained product with a magnet, and wash the product with hot water and methanol several times to obtain magnetic polydivinylbenzene-vinylbenzyl chloride copolymer microspheres (PDVB-VBC).

[0179] 2. Preparation of carboxyl-modified magnetic polymer microspheres:

[0180] Disperse 5 g of the magnetic polymer microspheres obtained in Step 1 in 60 mL of KOH (3 M) solution, react with stirring at 60 °C for 8 hours, and after the reaction is completed, wash with pure water several times until the pH = 7. Then further disperse it in 100 mL of NaClO (effective chlorine is 12%) solution, react with stirring at 60 °C for 1.5 h. After the reaction is completed, wash with pure water several times to obtain carboxyl-modified magnetic polymer microspheres.

[0181] 3. Preparation of magnetic polymer microspheres with doubled carboxyl density;

[0182] 3a) Take 0.5 g of the carboxyl-functionalized microspheres obtained in Step 2 after drying, disperse them in 50 mL of methanol, and add 15 mL of ethylenediamine, and react with stirring at room temperature for 1 day.

[0183] 3b) Wash the product obtained after the reaction in Step 3a) with pure water several times, then redisperse it in 50 mL of methanol, add 10 mL of methyl acrylate, and react with stirring at room temperature for 1 day under a nitrogen atmosphere.

[0184] Repeat Steps 3a) and 3b), alternately perform 3 - 5 times, and finally disperse the product - multi-ester-based microspheres in 60 mL of methanol solution containing 1.5 mM lithium hydroxide, react with stirring at room temperature for 12 h. After the reaction is completed, wash the product with methanol and pure water several times, and then disperse and store it in methanol to obtain magnetic polymer microspheres with doubled carboxyl density.

[0185] 4. Wash 2.5 g of the magnetic polymer microspheres with doubled carboxyl groups obtained in Step 3 three times with 1,2-dichloroethane, then disperse them in 30 mL of 1,2-dichloroethane, and stir and swell at room temperature for 0.5 hour. Add 2 mL of dimethoxymethane and 0.5 g of FeCl 3 , react under a nitrogen atmosphere, and stir and react at room temperature for 4 h. After the reaction is completed, wash with anhydrous methanol several times, and then magnetic microspheres with high specific surface area and high carboxyl density can be obtained.

[0186] Characterization method:

[0187] 1) Morphology analysis: Analyzed using an Olympus optical microscope. The sample was dispersed in methanol during measurement.

[0188] 2) Specific surface area analysis: Using Micromeritics ASAP 2460, the sample was degassed at 95 °C for 12 hours for activation.

[0189] 3) Magnetization intensity analysis: Using LakeShore7404, the test conditions were at room temperature within ±2T.

[0190] 4) Magnetic response time analysis: Using SEPMAG - A4000 MA211 magnetic separator, the test volume was 2 mL, and the dispersion solvent was pure water.

[0191] 5) Functional group density analysis: Using Metrohm 916 automatic potentiometric titrator, titrated with KOH solution.

[0192] Characterization results:

[0193] The microspheres are spherical in shape, and most of the particle sizes are distributed between 10 - 50 μm. They are superparamagnetic, and the saturation magnetization intensity is about 6.1 emu / g. The magnetic response time in pure water is about 15 s. At the same time, the specific surface area is about 700 m 2 / g, the pore size is mainly concentrated around 4 nm, and the carboxyl content on the surface is 0.61 mmol / g. It meets the requirements.

[0194] See Figure 1 , Figure 1 which is the morphology diagram of the magnetic microspheres with high specific surface area and high carboxyl density prepared by the present invention.

[0195] See Figure 2 , Figure 2 which is the saturation magnetization intensity data diagram of the magnetic microspheres with high specific surface area and high carboxyl density prepared by the present invention.

[0196] See Figure 3 , Figure 3 which is the characteristic diagram of the magnetic microspheres with high specific surface area and high carboxyl density prepared by the present invention. Among them, the upper figure is the nitrogen adsorption - desorption curve, and the lower figure is the pore size distribution diagram.

[0197] See Figure 4 , Figure 4 which is the magnetic response time curve diagram of the magnetic microspheres with high specific surface area and high carboxyl density prepared by the present invention.

[0198] See Figure 5 , Figure 5Potentiometric titration curve of magnetic microspheres with high specific surface area and high carboxyl density prepared for the present invention.

[0199] Application Example

[0200] Application Scheme 1: Applicable to four steroid hormones (17-hydroxyprogesterone 17-OHP, dihydrotestosterone DHT, androstenedione A4, testosterone T);

[0201] Application Scheme 2: Applicable to 3 catecholamine metabolites (metanephrine MN, normetanephrine NMN, 3-methoxytyramine 3-MT);

[0202] Application Scheme 3: Applicable to simultaneous extraction of four steroid hormones (17-hydroxyprogesterone 17-OHP, dihydrotestosterone DHT, androstenedione A4, testosterone T) and 3 catecholamine metabolites (metanephrine MN, normetanephrine NMN, 3-methoxytyramine 3-MT);

[0203] Application Scheme 4: Applicable to first extract four steroid hormones (17-hydroxyprogesterone 17-OHP, dihydrotestosterone DHT, androstenedione A4, testosterone T), and then extract 3 catecholamine metabolites (metanephrine MN, normetanephrine NMN, 3-methoxytyramine 3-MT).

[0204] Application Scheme 1

[0205] Multifunctional magnetic beads are applicable to five steroid hormones

[0206] 1. Component preparation:

[0207] Eluent: Take 5 mL of methanol and add 95 mL of water, vortex and mix well to prepare 5% methanol-water for standby;

[0208] Magnetic suspension: Disperse magnetic beads according to 1 g of magnetic beads requiring 10 mL of methanol. After magnetic absorption, remove the methanol and add 5% methanol-water to prepare a 5 mg / mL magnetic suspension;

[0209] Eluate: A methanol / acetonitrile mixed solution with a volume ratio of 50%.

[0210] 2. Pretreatment process of the kit

[0211] 1) Add 20 μL of internal standard working solution and 100 μL of magnetic suspension to 100 μL of serum, mix well, magnetic absorb for 1 min, and remove the supernatant;

[0212] 2) Add 200 μL of purified water, mix well, magnetic absorb for 1 min, and remove the supernatant;

[0213] 3) Add 200 μL of eluent, mix well, magnetic absorb for 1 min, and remove the supernatant;

[0214] 4) Add 100 μL of eluent, mix well, magnetically attract for 1 min, and transfer 80 μL of the supernatant to the sample vial;

[0215] 5) Add 80 μL of purified water, mix well and then load onto the instrument.

[0216] 3. Instrument detection parameters

[0217] Instrument model: AB SCIEX liquid chromatography-tandem mass spectrometry detection system 4500MD

[0218] Ionization mode: ESI+, curtain gas (CUR): 25 psi, collision gas (CAD): 7, ionization voltage (IS): 5000 V, temperature (TEM): 400 °C, nebulizing gas (GS1): 60 psi, auxiliary heating gas (GS2): 50 psi;

[0219] Ionization mode: ESI-, curtain gas (CUR): 20 psi, collision gas (CAD): 9, ionization voltage (IS): -3500 V, temperature (TEM): 400 °C, nebulizing gas (GS1): 45 psi, auxiliary heating gas (GS2): 60 psi;

[0220] Ion pair parameters are shown in Table 1. Table 1 shows the ion pair parameters in Application Scheme 1 of the present invention.

[0221] Table 1

[0222] Compound Q1 Q3 Dwell (ms) DP (V) EP (V) CE (V) CXP (V) 17-OHP 331.1 109.1 40 110 10 34 11 17-OHP-13C3 334.1 112.1 40 110 10 36 11 DHT 291.3 255.4 40 110 10 28 11 DHT-D3 294.4 258.3 40 110 10 23 11 A4 287.2 97.1 40 120 10 28 11 A4-13C3 290.4 100.1 40 120 10 30 11 T 289.1 97.0 40 100 10 30 11 T-13C3 292.1 100.1 40 100 10 30 11 DHEAS 367.1 97 40 -100 -10 -40 -10 DHEAS-D6 373.2 98 40 -100 -10 -60 -10

[0223] Chromatographic column type: Kinetex C18 2.1*500 mm 2.6 μm; column temperature: 40 °C;

[0224] Injection volume: 40 μL;

[0225] Mobile phase A: 2 mM ammonium formate aqueous solution; mobile phase B: 2 mM ammonium formate methanol.

[0226] See Table 2. Table 2 shows the liquid chromatography parameters in Application Scheme 1 of the present invention.

[0227] Table 2

[0228] Time (min) Flow rate (mL / min) A％ B％ 0 0.6 50 50 0.5 0.6 50 50 2.5 0.6 30 70 2.7 0.6 5 95 3.2 0.6 5 95 3.3 0.6 50 50 4.5 0.6 50 50

[0229] See Figure 6 , Figure 6 which is the chromatogram of 17-OHP and 17-OHP-13C3 in Application Scheme 1 of the present invention.

[0230] See Figure 7 , Figure 7 which is the chromatogram of DHT and DHT-D3 in Application Scheme 1 of the present invention.

[0231] See Figure 8 , Figure 8 which is the chromatogram of A4 and A4-13C3 in Application Solution 1 of the present invention.

[0232] See Figure 9 , Figure 9 which is the chromatogram of T and T-13C3 in Application Solution 1 of the present invention.

[0233] See Figure 10 , Figure 10 which is the chromatogram of DHEAS and DHEAS-D6 in Application Solution 1 of the present invention.

[0234] 4. Comparison with the extraction effect of commercial magnetic beads

[0235] The self-made multifunctional magnetic beads and commercial HLB magnetic beads (Boyun) were respectively prepared into the same concentration according to the preparation scheme of the magnetic suspension, and the same batch of samples were pretreated. The extraction peak areas are shown in Table 3. Table 3 shows the extraction peak areas of the multifunctional magnetic beads and commercial HLB magnetic beads in Application Solution 1.

[0236] Table 3

[0237] Analyte Multifunctional magnetic bead Area Commercial HLB magnetic bead Area Area deviation 17-OHP 5.39E+05 5.57E+05 -3.2％ DHT 2.80E+05 3.00E+05 -6.8％ A4 1.11E+06 1.09E+06 2.1％ T 1.70E+06 1.82E+06 -6.9％ DHEAS 1.20E+07 1.95E+07 -38.7％

[0238] The results show that the deviation of the extraction peak areas of the four substances is within 7%, meeting the detection requirements; although the extraction effect of DHEAS is relatively poor, the normal concentration of this substance in the sample is far higher than the detection limit of the instrument, and it still meets the detection requirements.

[0239] Application Solution 2

[0240] The multifunctional magnetic beads are applicable to three catecholamine metabolites

[0241] 1. Component preparation:

[0242] Eluent: Take 5 mL of methanol and add 95 mL of water, vortex and mix evenly to prepare 5% methanol-water for standby;

[0243] Magnetic suspension: According to 1 g of magnetic beads requires 10 mL of methanol to disperse the magnetic beads. After magnetic absorption, remove the methanol and add 5% methanol-water to prepare a 5 mg / mL magnetic suspension;

[0244] Eluate: A 2% formic acid / acetonitrile mixed solution by volume.

[0245] 2. Pretreatment process of the kit

[0246] 1) Add 20 μL of internal standard working solution and 100 μL of magnetic suspension to 400 μL of serum, mix evenly, magnetically absorb for 1 min, and remove the supernatant;

[0247] 2) Add 200 μL of purified water and mix well. Magnetically attract for 1 minute and remove the supernatant.

[0248] 3) Add 200 μL of eluent and mix well. Magnetically attract for 1 minute and remove the supernatant.

[0249] 4) Add 100 μL of elution solution and mix well. Magnetically attract for 1 minute and take the supernatant for on-machine detection.

[0250] 3. Instrument detection parameters

[0251] Instrument model: AB SCIEX liquid chromatography tandem mass spectrometry detection system 4500MD

[0252] Ionization mode: ESI+, curtain gas (CUR): 30 psi, collision gas (CAD): 7, ionization voltage (IS): 5500 V, temperature (TEM): 500 °C, nebulizing gas (GS1): 50 psi, auxiliary heating gas (GS2): 60 psi;

[0253] Ion pair parameters are shown in Table 4. Table 4 shows the ion pair parameters in Application Scheme II of the present invention.

[0254] Table 4

[0255] Compound Q1 Q3 Dwell (ms) DP (V) EP (V) CE (V) CXP (V) MN 180.1 148.1 25 90 10 25 11 MN-d3 183.1 151.1 25 80 10 25 11 NMN 166.1 134.1 25 85 10 23 11 NMN-d3 169.1 137.1 25 80 10 25 11 3-MT 168.1 119.1 25 50 10 26 11 3-MT-d4 172.1 123.1 25 55 10 16 11

[0256] Chromatographic column type: Phenomenex Hilic chromatographic column, 2.1×100 mm, 2.6 μm;

[0257] Column temperature: 35 °C; injection volume: 40 μL

[0258] Mobile phase A: Aqueous solution of 10 mM ammonium formate with pH 3; Mobile phase B: Acetonitrile. See Table 5. Table 5 shows the liquid chromatography parameters in Application Scheme II of the present invention.

[0259] Table 5

[0260] Time (min) Flow rate (mL / min) A％ B％ 1 0.45 0 100 3 0.45 30 70 3.5 0.45 40 60 3.6 0.45 40 60 5.0 0.45 0 100

[0261] See Figure 11 , Figure 11 which is the chromatogram of MN and MN-d3 in Application Scheme II of the present invention.

[0262] See Figure 12 , Figure 12 which is the chromatogram of NMN and NMN-d3 in Application Scheme II of the present invention.

[0263] See Figure 13 , Figure 13 which is the chromatogram of 3-MT and 3-MT-d4 in Application Scheme II of the present invention.

[0264] 4. Comparison with the extraction effect of commercial magnetic beads

[0265] The self-made multifunctional magnetic beads and commercial WCX magnetic beads (Boyun) were respectively prepared into the same concentration according to the preparation scheme of the magnetic suspension, and the same batch of samples were pretreated. The extraction peak areas are shown in Table 6. Table 6 shows the extraction peak areas of the multifunctional magnetic beads and commercial WCX magnetic beads in Application Scheme II.

[0266] Table 6

[0267] Analyte Multifunctional magnetic bead Area Commercial WCX magnetic bead Area Area deviation MN 2.70E+06 2.69E+06 0.6％ NMN 4.30E+05 4.82E+05 -10.7％ 3-MT 1.58E+06 1.89E+06 -15.0％

[0268] The results show that the deviation of the extraction peak areas of the three substances is within 15%, meeting the detection requirements.

[0269] Application Scheme III

[0270] The multifunctional magnetic beads are suitable for the combined detection of four steroid hormones and three catecholamine metabolites

[0271] 1. Component preparation:

[0272] Eluent: Take 5 mL of methanol and add it to 95 mL of water, vortex and mix well to prepare 5% methanol-water for standby;

[0273] Magnetic suspension: Disperse the magnetic beads according to 1 g of magnetic beads requiring 10 mL of methanol. After magnetic absorption, remove the methanol and add 5% methanol-water to prepare a 5 mg / mL magnetic suspension;

[0274] Elution solution: A 2% formic acid / acetonitrile mixed solution by volume.

[0275] 2. Pretreatment process of the kit

[0276] 1) Add 20 μL of the internal standard working solution and 100 μL of the magnetic suspension to 400 μL of serum, mix well, magnetically absorb for 1 min, and remove the supernatant;

[0277] 2) Add 200 μL of purified water, mix well, magnetically absorb for 1 min, and remove the supernatant;

[0278] 3) Add 200 μL of the eluent, mix well, magnetically absorb for 1 min, and remove the supernatant;

[0279] 4) Add 100 μL of the elution solution, mix well, magnetically absorb for 1 min, and take 70 μL of the supernatant for the detection of catecholamine metabolites.

[0280] 5) Take 20 μL of the supernatant, add 60 μL of the elution solution and 80 μL of purified water, mix well, and then perform the detection of four steroid hormones.

[0281] This pretreatment process is also applicable to the extraction of plasma samples.

[0282] 3. Comparison of two elution methods for four steroid hormones

[0283] The self-made multifunctional magnetic beads can be used to extract steroid hormones with common eluents, and are also compatible with the extraction of catecholamine items. Pretreat the same batch of samples, and the extraction peak areas are shown in Table 7. Table 7 shows the extraction peak areas of the multifunctional magnetic beads in Application Plan 3.

[0284] Table 7

[0285] Analyte Steroid hormone extraction Area Steroid hormone & catecholamine extraction Area Area deviation 17-OHP 5679 11475 102％ DHT 1921 2785 45％ A4 49350 72655 47％ T 23425 39865 70％

[0286] The results show that when the combined extraction method of steroid hormones & catecholamines is adopted, the extraction peak area of steroid hormones increases between 45% and 100%. It is speculated that the acidic environment has a certain improvement in the extraction of the four steroid hormones, meeting the detection requirements.

[0287] Application Plan 4

[0288] The multifunctional magnetic beads first extract four steroid hormones and then extract three catecholamine metabolites.

[0289] 1. Component preparation:

[0290] Eluent: Take 5 mL of methanol and add 95 mL of water, vortex and mix well to prepare 5% methanol-water for standby;

[0291] Magnetic suspension: Disperse the magnetic beads according to 1 g of magnetic beads requiring 10 mL of methanol. After magnetic absorption, remove the methanol and add 5% methanol-water to prepare a 5 mg / mL magnetic suspension;

[0292] Eluent 1: A 50% formic acid / acetonitrile mixture by volume.

[0293] Eluent 2: A 2% formic acid / acetonitrile mixture by volume.

[0294] 2. Pretreatment process of the kit

[0295] 1) Add 20 μL of internal standard working solution and 100 μL of magnetic suspension to 400 μL of serum, mix well, perform magnetic absorption for 1 min, and remove the supernatant;

[0296] 2) Add 200 μL of purified water, mix well, perform magnetic absorption for 1 min, and remove the supernatant;

[0297] 3) Add 200 μL of eluent, mix well, perform magnetic absorption for 1 min, and remove the supernatant;

[0298] 4) Add 100 μL of Eluent 1, mix well, perform magnetic absorption for 1 min, take 80 μL of the supernatant, and then add 80 μL of purified water, mix well, and perform the detection of the four steroid hormones.

[0299] 5) Add 100 μL of Eluent 2, mix well, perform magnetic absorption for 1 min, and take the supernatant for the detection of catecholamine metabolites.

[0300] 3. Comparison of Two Extraction Methods for Three Catecholamines

[0301] The self-made multifunctional magnetic beads can directly extract three catecholamines; they can also first perform steroid hormone elution and then perform secondary elution of catecholamine metabolites; for the same batch of samples, the extraction peak areas are shown in Table 8. Table 8 shows the extraction peak areas of the multifunctional magnetic beads in Application Scheme 4.

[0302] Table 8

[0303] Analyte Catecholamine metabolite direct extraction Area Catecholamine metabolite secondary extraction Area Area deviation MN 276563 240562 -13％ NMN 66604 57129 -14％ 3-MT 78636 71093 -10％

[0304] The results show that when the method of first extracting steroid hormones and then extracting catecholamine metabolites is adopted, it is speculated that the simple organic reagent in eluent 1 has a certain extraction effect on catecholamine metabolites, resulting in a certain loss, but the deviation is within 15%, meeting the requirements.

[0305] The above provides a detailed introduction to the present invention, especially relating to a synthesis method and application of a multifunctional magnetic bead. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention, including the best mode, and also enables any person skilled in the art to practice the present invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The protection scope of the present invention patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements that are not different from the literal description of the claims, or if they include equivalent structural elements that have no substantial difference from the literal description of the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A method for synthesizing magnetic beads, characterized in that: The following steps are involved: 1) mixing an aqueous dispersion of magnetic nanoparticles with oleic acid, reacting the mixture and then separating the mixture to obtain oleic acid-coated magnetic cores; 2) mixing the oleic acid-coated magnetic core obtained in the above step, the first polymerizable monomer, the second polymerizable monomer, the oil phase solvent and the initiator to obtain an oil phase mixed solution; 3) adding the oil phase mixed solution obtained in the above step into the polyvinyl alcohol aqueous dispersion to react, thereby obtaining magnetic polymer microspheres with the copolymer coated on the surface; 4) subjecting the magnetic polymer microspheres obtained in the above step to a first reaction with an alkaline solution, and then mixing with an oxidant to a second reaction to obtain carboxyl-modified magnetic polymer microspheres; 5) mixing the carboxyl-modified magnetic polymer microspheres obtained in the above step, the diamine compound and the first solvent to react, thereby obtaining amino-modified microspheres, and then mixing the amino-modified microspheres, the acrylate compound and the first solvent again to react, thereby obtaining polyester-based microspheres; 6) reacting the polyester-based microspheres obtained in the above step in a solvent containing a Lewis base to obtain microspheres with amplified carboxyl group density; 7) Under protective atmosphere and Lewis acid catalyst, the microspheres with amplified carboxyl density obtained in the above step, a cross-linking agent and a second solvent are mixed for cross-linking reaction to obtain magnetic beads.

2. The synthesis method according to claim 1, characterized in that The magnetic nanoparticles include Fe3O4 nanoparticles; The particle size of the magnetic nanoparticles is 5 to 20 nm; The pH value of the aqueous dispersion of the magnetic nanoparticles is 7 to 9; The mixing reaction in step 1) is specifically to drop oleic acid into the aqueous dispersion of magnetic nanoparticles for reaction; The magnetic polymer microspheres are specifically magnetic polymer microspheres with copolymers coated on the surface.

3. The synthesis method according to claim 1, characterized in that The mass ratio of the oleic acid-coated magnetic core to the first polymerizable monomer is (1-5):(3-6); The first polymerizable monomer includes divinylbenzene; The second polymerizable monomer includes vinylbenzyl chloride; The volume ratio of the first polymerizable monomer to the second polymerizable monomer is 1:(1-3); The initiator includes azobisisobutyronitrile; The mass ratio of the azobisisobutyronitrile to the first polymerizable monomer is 1:(1-15).

4. The synthesis method according to claim 1, characterized in that The oil phase solvent includes one or more of 1,2-dichloroethane, toluene, xylene and dichloromethane; The content of polyvinyl alcohol in the polyvinyl alcohol aqueous dispersion is 1wt% to 10wt%; The volume ratio of the oil phase mixed solution to the polyvinyl alcohol aqueous dispersion is 1:(2-6); The adding method includes dropwise addition; In the step 3), the reaction temperature is 60-100°C; In the step 3), the reaction time is 2 to 8 hours; The magnetic polymer microspheres include magnetic polydivinylbenzene-vinylbenzyl chloride copolymer microspheres.

5. The synthesis method according to claim 1, characterized in that The alkaline solution includes a sodium hydroxide solution; The temperature of the first reaction is 20 to 80°C; The first reaction time is 4 to 16 hours; The oxidant includes sodium hypochlorite; The temperature of the second reaction is 40 to 80°C; The second reaction time is 1 to 4 hours.

6. The synthesis method according to claim 1, characterized in that The step 4) may also be the following steps: 4') subjecting the magnetic polymer microspheres obtained in the above step, aromatic carboxylic acid monomers and alkaline solution to a nucleophilic substitution reaction to obtain carboxyl-modified magnetic polymer microspheres; The diamine compound includes one or more of ethylenediamine, propylenediamine and butylenediamine; The mass volume ratio of the magnetic polymer microspheres to the diamine compound is 1 g: (1-4) mL; The first solvent is selected from one or more of an alcohol solvent, an ether solvent, a ketone solvent, a nitrile solvent and pyridine; The mixing reaction time is 12 to 48 hours; The acrylic acid ester compound includes one or more of methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, n-butyl acrylate, tert-butyl acrylate, isobutyl acrylate, n-pentyl acrylate, isopentyl acrylate and hexyl acrylate; The mass volume ratio of the amino-modified microspheres to the acrylic ester compound is 1 g: (1-5) mL.

7. The synthesis method according to claim 1, characterized in that The time for the re-mixing reaction is 12 to 48 hours; The step 5) is specifically repeated multiple times; The number of times is 3 to 5 times; The Lewis base includes one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium oxide and calcium oxide; The Lewis base-containing solvent is selected from one or more of an alcohol solvent, an ether solvent, a ketone solvent, a nitrile solvent and pyridine; The mass ratio of the polyester-based microspheres to the Lewis base is (1-7):1; The Lewis acid catalyst includes one or more of ferric chloride, zinc chloride and aluminum chloride.

8. The synthesis method according to claim 1, characterized in that The mixing in step 7) is specifically as follows: firstly, the microspheres with amplified carboxyl density are mixed with the second solvent to swell, and then, after swelling, they are mixed again with the crosslinking agent and the Lewis acid catalyst to perform a crosslinking reaction; The swelling time is 0.5 to 2 hours; The mass ratio of the carboxyl density-enlarged microspheres to the Lewis acid catalyst is (1-7):1; The cross-linking agent includes dimethoxymethane; The mass ratio of the carboxyl density-amplified microspheres to the cross-linking agent is (1-5):1; The second solvent includes one or more of 1,2-dichloroethane, toluene, N,N-dimethylformamide, dimethyl sulfoxide and dichloromethane; The cross-linking reaction time is 2 to 48 hours.

9. The synthesis method according to claim 1, characterized in that The magnetic beads are specifically surface porous magnetic microspheres with high specific surface area and high carboxyl density; The particle size of the magnetic beads is 1 to 100 μm; The magnetic beads have superparamagnetism; The specific surface area of ​​the magnetic beads is ≥200m 2 / g; At least 80% of the magnetic beads have a pore size of ≤5 nm; The carboxyl content on the surface of the magnetic beads is 0.3 to 2 mmol / g; The saturation magnetization intensity of the magnetic beads is ≥4emu / g.

10. Use of the magnetic beads prepared by the synthesis method according to any one of claims 1 to 9 in extracting and / or detecting steroid hormones and / or catecholamine metabolites.

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