Solid-phase extraction column filler, and preparation method therefor and use thereof

Abstract

The present invention relates to a solid-phase extraction column filler. The solid-phase extraction column filler comprises: a microsphere body formed by copolymerization of several reaction monomers and several crosslinking monomers, and several graft monomers grafted on the surface of the microsphere body, wherein the reaction monomers are selected from acrylic acid; the cross-linking monomers are selected from at least one of divinyl benzene, diethylene glycol diacrylate, triethylene glycol diacrylate, and trimethylolpropane trimethacrylate; and the graft monomers are selected from heparin. According to the preparation method of the present invention, the acrylic acid is used as the reaction monomers and is copolymerized with the crosslinking monomers to generate the microsphere body; and then condensation grafting is carried out by means of a carboxyl functional group on the acrylic acid and an amino functional group of the heparin, such that preparation of the solid-phase extraction column filler is achieved, and the carboxyl functional group having electronegativity, a sulfonic acid group functional group of the heparin and the carboxyl functional group of the heparin can synergistically adsorb lactoferrin.

Description

A solid phase extraction column filler and its preparation method and application Technical Field

[0001] The present invention relates to the field of biochemical detection technology, and in particular to a solid phase extraction column filler and a preparation method and application thereof. Background Art

[0002] Heparin is a natural mucopolysaccharide sulfate, a linear polymer composed of alternating glucosamine, L-iduronide, N-acetylglucosamine, and D-glucuronic acid. The secondary amine and hydroxyl groups within the glucosamine backbone of heparin possess active hydrogens capable of forming hydrogen bonds with specific biomolecules. Furthermore, the sulfonic acid and carboxyl groups within the structural units of the heparin molecule impart a strong negative charge to heparin, enabling it to form ionic bonds with other positively charged groups. These two modes of action give heparin an affinity for a variety of proteins.

[0003] Existing heparin affinity columns typically use agarose gel as a solid-phase carrier, coupled to heparin to form an immunosorbent. However, agarose gel suffers from uneven particle size and poor mechanical strength, limiting its application in solid-phase extraction columns. Furthermore, the silanol groups in silica-based heparin affinity columns may interfere with the interaction between the heparin column and lactoferrin. In comparison, synthetic polymers offer a wider range of acid and alkali resistance, higher mechanical strength, and improved chemical stability.

[0004] Therefore, there is an urgent need for a solid phase extraction column filler and a preparation method and application thereof.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a solid phase extraction column filler and a preparation method and application thereof in view of the deficiencies in the prior art.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention is to provide a solid phase extraction column filler, comprising: a microsphere body formed by copolymerization of a plurality of reaction monomers and a plurality of cross-linking monomers, and a plurality of grafting monomers grafted onto the surface of the microsphere body; wherein,

[0009] The reactive monomer is selected from: acrylic acid;

[0010] The cross-linking monomer is selected from at least one of divinylbenzene, diethylene glycol diacrylate, triethylene glycol diacrylate, or trimethylolpropane trimethacrylate;

[0011] The grafting monomer is selected from the group consisting of: heparin.

[0012] Preferably, the particle size of the solid phase extraction column filler is 65 μm-250 μm; the specific surface area of ​​the solid phase extraction column filler is 200 m 2 / g-500m 2 / g; the pore volume of the solid phase extraction column filler is 0.80mL / g-2.50mL / g; the pore size of the solid phase extraction column filler is The cation exchange capacity of the solid phase extraction column filler is 0.5 mmol / g-3.1 mmol / g.

[0013] The second aspect of the present invention is to provide a method for preparing a solid phase extraction column filler, comprising the steps of:

[0014] S0-0, providing a reaction container;

[0015] S0-1. Providing a dispersion solution, wherein the dispersion solution comprises: deionized water and a dispersant dissolved in the deionized water;

[0016] S0-2. Providing a copolymerization solution, wherein the copolymerization solution includes: a first initiator, a first porogen, a reactive monomer, and a cross-linking monomer;

[0017] S0-3, providing a grafting solution, wherein the grafting solution includes: a second initiator, a second porogen, and a grafting monomer;

[0018] S1, transferring the dispersed solution into the reaction vessel, transferring the copolymerization solution into the reaction vessel under stirring, stirring for a first time, gradually heating to a first temperature, and copolymerizing for a second time to obtain a microsphere body;

[0019] S2, transferring a portion of the grafting solution to the reaction container, and uniformly transferring the catalyst to the reaction container, carrying out the grafting reaction for a third time, gradually raising the temperature to a second temperature, transferring another portion of the grafting solution to the reaction container, and carrying out the grafting reaction for a fourth time, to obtain the microsphere body grafted with the grafting monomer;

[0020] Wherein, the reactive monomer is selected from: acrylic acid;

[0021] Wherein, the cross-linking monomer is selected from: divinylbenzene, diethylene glycol diacrylate, triethylene glycol diacrylate, trimethylolpropane trimethacrylate;

[0022] Wherein, the grafting monomer is selected from: heparin.

[0023] Preferably, the dispersant is selected from at least one of polyvinyl pyrrolidone, sodium lauryl sulfate, hydroxypropyl methylcellulose, sodium lignin sulfonate, polyphosphate, or polycarboxylate.

[0024] Preferably, the mass ratio of the dispersant, the deionized water, and the cross-linking monomer is (2-3): (200-450): (10-35).

[0025] Preferably, the first initiator and the second initiator are independently selected from at least one of benzoyl peroxide, azobisisobutyronitrile, azobisdimethylvaleronitrile, or tert-butylhydroxyperoxide.

[0026] Preferably, the first porogen and the second porogen are independently selected from at least one of mesitylene, toluene, n-heptane, cyclohexanol, polystyrene, and polyacrylate.

[0027] Preferably, the mass ratio of the first initiator, the first porogen, the reactive monomer, and the cross-linking monomer is (0.1-0.3): (2-5): (0.2-0.5): (1-3).

[0028] Preferably, the mass ratio of the second initiator, the second porogen, the grafting monomer, and the cross-linking monomer is (0.1-0.35): (15-35): (5-17.5): (10-70).

[0029] Preferably, the molecular weight of the heparin is 3000-5000 Daltons.

[0030] Preferably, the stirring conditions include: 200-600 rpm.

[0031] Preferably, the first time is 0.5-1.5 hours.

[0032] Preferably, the first temperature is 40°C-60°C.

[0033] Preferably, the rate of heating to the first temperature is 2°C / min-5°C / min.

[0034] Preferably, the second time is 1-3 hours.

[0035] Preferably, the mass ratio of the one part of the grafting solution to the other part of the grafting solution is (1-2):(1-3).

[0036] Preferably, the catalyst is selected from: 4-(4,6-dioxo-1,3,5-triazin-2-yl)-4-methyl-2-oxazolyl-5-oxo-2-chlorophenyl-trifluoroacetic anhydride.

[0037] Preferably, the mass ratio of the catalyst to the cross-linking monomer is 1:(10-30).

[0038] Preferably, the rate at which the catalyst is transferred into the reaction vessel is 1.5 g / min to 2 g / min.

[0039] Preferably, the third time is 0.5-1.5 hours.

[0040] Preferably, the second temperature is 60°C-70°C.

[0041] Preferably, the rate of heating to the second temperature is 2°C / min-5°C / min.

[0042] Preferably, the fourth time is 1-3 hours.

[0043] Preferably, the steps further include:

[0044] S3. Spray-drying the microsphere body grafted with the grafted monomer to obtain the solid phase extraction column filler.

[0045] The third aspect of the present invention is to provide a use of a solid phase extraction column filler in detecting lactoferrin. The solid phase extraction column filler is as described above, or the solid phase extraction column filler is prepared by the preparation method as described above.

[0046] The fourth aspect of the present invention is to provide a solid phase extraction column, comprising: the solid phase extraction column filler as described above, or the solid phase extraction column filler prepared by the preparation method as described above.

[0047] The fifth aspect of the present invention is to provide an application of a solid phase extraction column in detecting lactoferrin, wherein the solid phase extraction column is as described above.

[0048] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0049] The solid-phase extraction column packing of the present invention has excellent physical properties, including: homogeneous particle size, large specific surface area, and uniform pore size. The cation exchange capacity can be flexibly adjusted within the range of 0.5 mmol / g to 3.1 mmol / g to accommodate different concentrations of the target substance. The adsorption recovery efficiency of lactoferrin is extremely high, reaching over 97%.

[0050] The preparation method of the present invention uses acrylic acid as a reaction monomer and copolymerizes it with a cross-linking monomer to form a microsphere main body; then, the carboxyl functional group on the acrylic acid is condensed and grafted with the amino functional group of heparin to achieve the preparation of a solid phase extraction column filler. The electronegative carboxyl functional group, the sulfonic acid functional group of heparin, and the carboxyl functional group of heparin can synergistically adsorb lactoferrin.

[0051] In addition, the preparation method of the present invention also adopts secondary grafting, which effectively increases the grafting amount of heparin. The process is simple and easy to operate, which is conducive to the implementation of industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1( a ) is a scanning electron microscopy image of the solid phase extraction column filler prepared in Example 3 at 2000 times magnification;

[0053] FIG1( b ) is a scanning electron microscopy result of the solid phase extraction column filler prepared in Example 3 at 10,000 times magnification;

[0054] FIG2 is a particle size test result of the solid phase extraction column filler prepared in Example 3;

[0055] FIG3 is a liquid chromatography result of standard substance in the lactoferrin recovery detection;

[0056] FIG4 is the liquid chromatography result corresponding to Example 3 in the detection of lactoferrin recovery rate. DETAILED DESCRIPTION

[0057] This embodiment provides a solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0058] The solid phase extraction column filler comprises: a microsphere body formed by copolymerization of a plurality of reaction monomers and a plurality of cross-linking monomers, and a plurality of grafting monomers grafted onto the surface of the microsphere body;

[0059] The preparation method of the solid phase extraction column filler comprises:

[0060] S0-0, providing a reaction container;

[0061] Wherein, in some preferred embodiments, the reaction vessel is a 50L reactor;

[0062] S0-1. Providing a dispersion solution, wherein the dispersion solution comprises: deionized water and a dispersant dissolved in the deionized water;

[0063] Wherein, the dispersant is selected from at least one of polyvinyl pyrrolidone, sodium lauryl sulfate, hydroxypropyl methylcellulose, sodium lignin sulfonate, polyphosphate, or polycarboxylate;

[0064] Wherein, the mass ratio of the dispersant, the deionized water, and the cross-linking monomer described later is (2-3): (200-450): (10-35);

[0065] S0-2. Providing a copolymerization solution, wherein the copolymerization solution includes: a first initiator, a first porogen, a reactive monomer, and a cross-linking monomer;

[0066] Wherein, the first initiator is selected from at least one of benzoyl peroxide, azobisisobutyronitrile, azobisdimethylvaleronitrile, or tert-butylhydroxyperoxide;

[0067] Wherein, the first porogen is selected from at least one of mesitylene, toluene, n-heptane, cyclohexanol, polystyrene, or polyacrylate;

[0068] Wherein, the reactive monomer is selected from: acrylic acid;

[0069] Wherein, the cross-linking monomer is selected from at least one of divinylbenzene, diethylene glycol diacrylate, triethylene glycol diacrylate, or trimethylolpropane trimethacrylate;

[0070] Wherein, the mass ratio of the first initiator, the first porogen, the reactive monomer, and the cross-linking monomer is (0.1-0.3): (2-5): (0.2-0.5): (1-3);

[0071] S0-3, providing a grafting solution, wherein the grafting solution includes: a second initiator, a second porogen, and a grafting monomer;

[0072] Wherein, the second initiator is selected from at least one of benzoyl peroxide, azobisisobutyronitrile, azobisdimethylvaleronitrile, or tert-butylhydroxyperoxide;

[0073] Wherein, the second porogen is selected from at least one of mesitylene, toluene, n-heptane, cyclohexanol, polystyrene, or polyacrylate;

[0074] Wherein, the grafting monomer is selected from: heparin;

[0075] Wherein, the molecular weight of the heparin is 3000-5000 Daltons;

[0076] Wherein, the mass ratio of the second initiator, the second porogen, the grafting monomer, and the cross-linking monomer is (0.1-0.35): (15-35): (5-17.5): (10-70);

[0077] S1. Transfer the dispersed solution to the reaction vessel, transfer the copolymerization solution to the reaction vessel under stirring conditions of 200-600 rpm, stir for 0.5-1.5 hours, gradually heat to 40-60°C at a heating rate of 2°C / min-5°C / min, and copolymerize for 1-3 hours to obtain a microsphere body;

[0078] Among them, in some preferred embodiments, the stirring conditions further include: using a polytetrafluoroethylene double-layer blade;

[0079] In some more preferred embodiments, the ratio of the width of the polytetrafluoroethylene double-layer blade to the diameter of the reactor is 1:6;

[0080] S2, transferring a portion of the grafting solution to the reaction vessel, and uniformly transferring the catalyst to the reaction vessel at a rate of 1.5 g / min-2 g / min, after the grafting reaction for 0.5-1.5 hours, gradually heating the temperature to 60° C.-70° C. at a heating rate of 2° C. / min-5° C. / min, transferring another portion of the grafting solution to the reaction vessel, and grafting the reaction for 1-3 hours to obtain the microsphere body grafted with the grafting monomer;

[0081] wherein the mass ratio of the one portion of the grafting solution to the other portion of the grafting solution is (1-2):(1-3);

[0082] Wherein, the catalyst is selected from: 4-(4,6-dioxo-1,3,5-triazine-2-yl)-4-methyl-2-oxazolyl-5-oxo-2-chlorophenyl-trifluoroacetic anhydride;

[0083] Wherein, the mass ratio of the catalyst to the cross-linking monomer is 1:(10-30);

[0084] S3. Spray-drying the microsphere body grafted with the grafted monomer to obtain the solid phase extraction column filler.

[0085] This embodiment also provides a solid phase extraction column and its application in detecting lactoferrin;

[0086] The solid phase extraction column comprises: the solid phase extraction column filler as described above.

[0087] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0088] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0089] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0090] Example 1

[0091] This embodiment provides a specific solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0092] The solid phase extraction column filler comprises: a microsphere main body formed by copolymerization of a plurality of acrylic acid (reactive monomer) and a plurality of triethylene glycol diacrylate (crosslinking monomer), and a plurality of heparin (grafting monomer) grafted onto the surface of the microsphere main body;

[0093] The preparation method of the solid phase extraction column filler comprises:

[0094] S0-0, provide a 50L reactor, using polytetrafluoroethylene double-layer blades for stirring;

[0095] S0-1. Providing a dispersion solution, the dispersion solution comprising: 20 L of deionized water and 150 g of hydroxypropyl methylcellulose (dispersant) dissolved in the deionized water;

[0096] S0-2. Providing a copolymerization solution, wherein the copolymerization solution comprises: 100 g of benzoyl peroxide (first initiator), 2000 g of toluene (first porogen), 200 g of acrylic acid (reactive monomer), and 1000 g of triethylene glycol diacrylate (crosslinking monomer);

[0097] S0-3. Providing a grafting solution, wherein the grafting solution comprises: 20 g of benzoyl peroxide (second initiator), 3000 g of mesitylene (second porogen), and 1000 g of heparin (grafting monomer);

[0098] Wherein, the molecular weight of the heparin is 3000-5000 Daltons;

[0099] S1, transferring the dispersed solution to the reaction vessel, and transferring the copolymerization solution to the reaction vessel under stirring at 400 rpm, stirring for 1 hour, gradually heating to 50° C. at a heating rate of 2° C. / min, and copolymerizing for 2 hours to obtain a microsphere body;

[0100] S2, transferring half of the grafting solution to the reaction container, and uniformly transferring 50 g of 4-(4,6-dioxo-1,3,5-triazine-2-yl)-4-methyl-2-oxazolyl-5-oxo-2-chlorophenyl-trifluoroacetic anhydride (catalyst) to the reaction container within 30 minutes. After grafting reaction for 1 hour, gradually heating the mixture to 65° C. at a heating rate of 2° C. / min, transferring the other half of the grafting solution to the reaction container, and grafting reaction for 2 hours to obtain the microsphere body grafted with the grafting monomer;

[0101] S3. Spray-drying the microsphere body grafted with the grafted monomer to obtain the solid phase extraction column filler.

[0102] Example 2

[0103] This embodiment provides a specific solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0104] The solid phase extraction column filler comprises: a microsphere main body formed by copolymerization of a plurality of acrylic acids (reactive monomers) and a plurality of divinylbenzenes (crosslinking monomers), and a plurality of heparins (grafting monomers) grafted onto the surface of the microsphere main body;

[0105] The preparation method of the solid phase extraction column filler comprises:

[0106] S0-0, provide a 50L reactor, using polytetrafluoroethylene double-layer blades for stirring;

[0107] S0-1. Providing a dispersion solution, the dispersion solution comprising: 22.5 L of deionized water, 100 g of polyvinyl pyrrolidone (a first dispersant) dissolved in the deionized water, and 50 g of sodium lauryl sulfate (a second dispersant) dissolved in the deionized water;

[0108] S0-2. Providing a copolymerization solution, the copolymerization solution comprising: 175 g of azobisisobutyronitrile (first initiator), 2916.7 g of mesitylene (first porogen), 291.7 g of acrylic acid (reactive monomer), and 1750 g of divinylbenzene (crosslinking monomer);

[0109] S0-3. Providing a grafting solution, wherein the grafting solution comprises: 17.5 g of benzoyl peroxide (second initiator), 1750 g of mesitylene (second porogen), and 875 g of heparin (grafting monomer);

[0110] Wherein, the molecular weight of the heparin is 3000-5000 Daltons;

[0111] S1, transferring the dispersed solution to the reaction vessel, and transferring the copolymerization solution to the reaction vessel under stirring at 400 rpm, stirring for 1 hour, gradually heating to 50° C. at a heating rate of 3° C. / min, and copolymerizing for 2 hours to obtain a microsphere body;

[0112] S2, transferring half of the grafting solution to the reaction vessel, and uniformly transferring 87.5 g of 4-(4,6-dioxo-1,3,5-triazine-2-yl)-4-methyl-2-oxazolyl-5-oxo-2-chlorophenyl-trifluoroacetic anhydride (catalyst) to the reaction vessel within 45 minutes. After grafting for 1 hour, gradually heating the mixture to 65° C. at a heating rate of 3° C. / min, transferring the other half of the grafting solution to the reaction vessel, and grafting for 2 hours to obtain the microsphere body grafted with the grafting monomer;

[0113] S3. Spray-drying the microsphere body grafted with the grafted monomer to obtain the solid phase extraction column filler.

[0114] Example 3

[0115] This embodiment provides a specific solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0116] The solid phase extraction column filler comprises: a microsphere main body formed by copolymerization of a plurality of acrylic acid (reactive monomer) and a plurality of triethylene glycol diacrylate (crosslinking monomer), and a plurality of heparin (grafting monomer) grafted onto the surface of the microsphere main body;

[0117] The preparation method of the solid phase extraction column filler comprises:

[0118] S0-0, provide a 50L reactor, using polytetrafluoroethylene double-layer blades for stirring;

[0119] S0-1. Providing a dispersion solution, the dispersion solution comprising: 21 L of deionized water, 100 g of polyvinyl pyrrolidone (a first dispersant) dissolved in the deionized water, and 100 g of sodium lauryl sulfate (a second dispersant) dissolved in the deionized water;

[0120] S0-2. Providing a copolymerization solution, the copolymerization solution comprising: 200 g of benzoyl peroxide (first initiator), 3000 g of mesitylene (first porogen), 280 g of acrylic acid (reactive monomer), and 2000 g of triethylene glycol diacrylate (crosslinking monomer);

[0121] S0-3. Providing a grafting solution, wherein the grafting solution comprises: 20 g of benzoyl peroxide (second initiator), 3100 g of mesitylene (second porogen), and 1000 g of heparin (grafting monomer);

[0122] Wherein, the molecular weight of the heparin is 3000-5000 Daltons;

[0123] S1, transferring the dispersed solution to the reaction vessel, and transferring the copolymerization solution to the reaction vessel under stirring at 400 rpm, stirring for 1 hour, gradually heating to 50° C. at a heating rate of 2° C. / min, and copolymerizing for 2 hours to obtain a microsphere body;

[0124] S2, transferring half of the grafting solution to the reaction vessel, and uniformly transferring 100 g of 4-(4,6-dioxo-1,3,5-triazine-2-yl)-4-methyl-2-oxazolyl-5-oxo-2-chlorophenyl-trifluoroacetic anhydride (catalyst) to the reaction vessel within 60 minutes. After grafting reaction for 1 hour, gradually heating the mixture to 65° C. at a heating rate of 2° C. / min, transferring the other half of the grafting solution to the reaction vessel, and grafting reaction for 2 hours to obtain the microsphere body grafted with the grafting monomer;

[0125] S3, spray drying the microsphere body grafted with the grafted monomer to obtain the solid phase extraction column filler;

[0126] The scanning electron microscopy results of the solid phase extraction column filler are shown in FIG1 , wherein FIG1( a ) is the scanning electron microscopy result at 2000 times magnification, and FIG1( b ) is the scanning electron microscopy result at 10000 times magnification.

[0127] Example 4

[0128] This embodiment provides a specific solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0129] The solid phase extraction column filler comprises: a microsphere main body formed by copolymerization of a plurality of acrylic acid (reactive monomer) and a plurality of triethylene glycol diacrylate (crosslinking monomer), and a plurality of heparin (grafting monomer) grafted onto the surface of the microsphere main body;

[0130] The preparation method of the solid phase extraction column filler comprises:

[0131] S0-0, provide a 50L reactor, using polytetrafluoroethylene double-layer blades for stirring;

[0132] S0-1. Providing a dispersion solution, the dispersion solution comprising: 21 L of deionized water and 100 g of polyvinyl pyrrolidone (dispersant) dissolved in the deionized water;

[0133] S0-2. Providing a copolymerization solution, the copolymerization solution comprising: 200 g of benzoyl peroxide (first initiator), 3000 g of mesitylene (first porogen), 280 g of acrylic acid (reactive monomer), and 2000 g of triethylene glycol diacrylate (crosslinking monomer);

[0134] S0-3. Providing a grafting solution, wherein the grafting solution comprises: 20 g of benzoyl peroxide (second initiator), 3100 g of mesitylene (second porogen), and 1000 g of heparin (grafting monomer);

[0135] Wherein, the molecular weight of the heparin is 3000-5000 Daltons;

[0136] S1, transferring the dispersed solution to the reaction vessel, and transferring the copolymerization solution to the reaction vessel under stirring at 400 rpm, stirring for 1 hour, gradually heating to 50° C. at a heating rate of 2° C. / min, and copolymerizing for 2 hours to obtain a microsphere body;

[0137] S2, transferring half of the grafting solution to the reaction vessel, and uniformly transferring 100 g of 4-(4,6-dioxo-1,3,5-triazine-2-yl)-4-methyl-2-oxazolyl-5-oxo-2-chlorophenyl-trifluoroacetic anhydride (catalyst) to the reaction vessel within 60 minutes. After grafting reaction for 1 hour, gradually heating the mixture to 65° C. at a heating rate of 2° C. / min, transferring the other half of the grafting solution to the reaction vessel, and grafting reaction for 2 hours to obtain the microsphere body grafted with the grafting monomer;

[0138] S3. Spray-drying the microsphere body grafted with the grafted monomer to obtain the solid phase extraction column filler.

[0139] Comparative Example 1

[0140] This comparative example provides another solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0141] The solid phase extraction column filler comprises: a microsphere main body formed by copolymerization of a plurality of acrylic acid (reactive monomer) and a plurality of triethylene glycol diacrylate (crosslinking monomer), and a plurality of heparin (grafting monomer) grafted onto the surface of the microsphere main body;

[0142] The preparation method of the solid phase extraction column filler comprises:

[0143] S0-0, provide a 50L reactor, using polytetrafluoroethylene double-layer blades for stirring;

[0144] S0-1. Providing a dispersion solution, the dispersion solution comprising: 21 L of deionized water, 100 g of polyvinyl pyrrolidone (a first dispersant) dissolved in the deionized water, and 100 g of sodium lauryl sulfate (a second dispersant) dissolved in the deionized water;

[0145] S0-2. Providing a copolymerization solution, the copolymerization solution comprising: 200 g of benzoyl peroxide (first initiator), 3000 g of mesitylene (first porogen), 280 g of acrylic acid (reactive monomer), and 2000 g of triethylene glycol diacrylate (crosslinking monomer);

[0146] S0-3. Providing a grafting solution, wherein the grafting solution comprises: 10 g of benzoyl peroxide (second initiator), 1550 g of mesitylene (second porogen), and 500 g of heparin (grafting monomer);

[0147] Wherein, the molecular weight of the heparin is 3000-5000 Daltons;

[0148] S1, transferring the dispersed solution to the reaction vessel, and transferring the copolymerization solution to the reaction vessel under stirring at 400 rpm, stirring for 1 hour, gradually heating to 50° C. at a heating rate of 2° C. / min, and copolymerizing for 2 hours to obtain a microsphere body;

[0149] S2, transferring the grafting solution to the reaction vessel, and uniformly transferring 100 g of 4-(4,6-dioxo-1,3,5-triazine-2-yl)-4-methyl-2-oxazolyl-5-oxo-2-chlorophenyl-trifluoroacetic anhydride (catalyst) to the reaction vessel within 60 minutes, and after grafting reaction for 1 hour, gradually heating the mixture to 65° C. at a heating rate of 2° C. / min, and grafting reaction for 2 hours to obtain the microsphere body grafted with the grafting monomer;

[0150] S3. Spray-drying the microsphere body grafted with the grafted monomer to obtain the solid phase extraction column filler.

[0151] Comparative Example 2

[0152] This comparative example provides another solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0153] The solid phase extraction column filler comprises: a microsphere body formed by copolymerization of a plurality of acrylic acids (reaction monomers) and a plurality of triethylene glycol diacrylates (crosslinking monomers);

[0154] The preparation method of the solid phase extraction column filler comprises:

[0155] S0-0, provide a 50L reactor, using polytetrafluoroethylene double-layer blades for stirring;

[0156] S0-1. Providing a dispersion solution, the dispersion solution comprising: 21 L of deionized water, 100 g of polyvinyl pyrrolidone (a first dispersant) dissolved in the deionized water, and 100 g of sodium lauryl sulfate (a second dispersant) dissolved in the deionized water;

[0157] S0-2. Providing a copolymerization solution, the copolymerization solution comprising: 200 g of benzoyl peroxide (first initiator), 3000 g of mesitylene (first porogen), 280 g of acrylic acid (reactive monomer), and 2000 g of triethylene glycol diacrylate (crosslinking monomer);

[0158] S1, transferring the dispersed solution to the reaction vessel, and transferring the copolymerization solution to the reaction vessel under stirring at 400 rpm, stirring for 1 hour, gradually heating to 50° C. at a heating rate of 2° C. / min, and copolymerizing for 2 hours to obtain a microsphere body;

[0159] S3, spray-drying the microsphere body to obtain the solid phase extraction column filler.

[0160] Comparative Example 3

[0161] This comparative example provides another solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0162] The solid phase extraction column filler comprises: a microsphere body formed by polymerization of a plurality of triethylene glycol diacrylate (cross-linking monomer);

[0163] The preparation method of the solid phase extraction column filler comprises:

[0164] S0-0, provide a 50L reactor, using polytetrafluoroethylene double-layer blades for stirring;

[0165] S0-1. Providing a dispersion solution, the dispersion solution comprising: 21 L of deionized water, 100 g of polyvinyl pyrrolidone (a first dispersant) dissolved in the deionized water, and 100 g of sodium lauryl sulfate (a second dispersant) dissolved in the deionized water;

[0166] S0-2. Providing a polymerization solution, wherein the polymerization solution comprises: 200 g of benzoyl peroxide (first initiator), 3000 g of mesitylene (first porogen), and 2000 g of triethylene glycol diacrylate (crosslinking monomer);

[0167] S0-3. Providing a grafting solution, wherein the grafting solution comprises: 20 g of benzoyl peroxide (second initiator), 3100 g of mesitylene (second porogen), and 1000 g of heparin (grafting monomer);

[0168] Wherein, the molecular weight of the heparin is 3000-5000 Daltons;

[0169] S1, transferring the dispersed solution to the reaction vessel, and transferring the polymerized solution to the reaction vessel under stirring at 400 rpm, stirring for 1 hour, gradually heating to 50° C. at a heating rate of 2° C. / min, and performing polymerization reaction for 2 hours to obtain a microsphere body;

[0170] S2, transferring half of the grafting solution to the reaction vessel, and after grafting reaction for 1 hour, gradually heating the temperature to 65° C. at a heating rate of 2° C. / min, and transferring the other half of the grafting solution to the reaction vessel, and grafting reaction for 2 hours. Since the surface of the microsphere body does not contain carboxyl functional groups, the grafting monomer cannot be covalently grafted to the surface of the microsphere body;

[0171] S3, spray-drying the microsphere body to obtain the solid phase extraction column filler.

[0172] Comparative Example 4

[0173] This comparative example provides another solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0174] The solid phase extraction column is: HiTrap TM Heparin HP, supplied by GE, is available in 5 mL format.

[0175] Note: The filler carrier of the solid phase extraction column in this comparative example is agarose, and the flow rate through the column is too slow, and external pressure is required to allow the sample liquid to pass through the solid phase extraction column and enter the collection tube.

[0176] Comparative Example 5

[0177] This comparative example provides another solid phase extraction column filler and its preparation method and application in detecting lactoferrin;

[0178] The solid phase extraction column is: SelectCor eTM Heparin, provided by sodium spectroscopy, is available in 1 mL quantities.

[0179] Note: The filler carrier of the solid phase extraction column in this comparative example is polyacrylate microspheres, which are expensive.

[0180] Detection Example

[0181] ① Test the ion exchange capacity of Examples 1-4 and Comparative Examples 1-5

[0182] Take two portions of solid-phase extraction column filler, 0.9g-1.1g each (accurate to 0.0001g), and place them separately in dry stoppered Erlenmeyer flasks; use a pipette to add 100.00mL of 0.1mol / L sodium hydroxide standard solution, shake well, cover the bottle tightly, and soak in a 60℃ water bath for 3 hours. Remove and cool to room temperature; use a pipette to remove 25mL of the soaking solution from the stoppered Erlenmeyer flask (do not aspirate the solid-phase extraction column filler) and place it in an Erlenmeyer flask, add 50mL of pure water and 3 drops of mixed indicator solution; titrate with 0.1mol / L hydrochloric acid standard solution until the color becomes slightly purple-red and does not fade for 15 seconds, which is the endpoint. At the same time, perform a blank test;

[0183] Wherein, Q is the total exchange capacity of the cation exchange resin on a wet basis, mmol / g;

[0184] c(HCl) is the concentration of hydrochloric acid standard solution, mol / L;

[0185] V1 is the volume of hydrochloric acid standard solution consumed in titrating the soaking solution, mL;

[0186] V2 is the volume of hydrochloric acid standard solution consumed in the blank test, mL;

[0187] M is the mass of the sample, g;

[0188] The arithmetic mean of the results of the two solid phase extraction column packings was calculated, and the results were rounded to two decimal places. The results are shown in Table 1;

[0189] ②Particle size test of Examples 1-4 and Comparative Examples 1-6

[0190] The analysis was performed using a Malvern Laser Particle Size Analyzer 2000. The dispersion solvent of the solid phase extraction column packing was deionized water, the refractive index of the solid phase extraction column packing was 1.520, the absorbance of the solid phase extraction column packing was 0.1, and the analysis mode was Single Narrow Mode. The results of Example 3 are shown in FIG2 , and the remaining results are shown in Table 1.

[0191] ③ Test the recovery rate of lactoferrin in Examples 1-4 and Comparative Examples 1-6

[0192] Take the solid phase extraction column filler and pack it into the column. The capacity of the solid phase extraction column is 3 mL, and each solid phase extraction column is loaded with 60 mg of solid phase extraction column filler. The specific steps include:

[0193] P0, matrix pretreatment: weigh 5.0 g of infant formula sample (accurate to 0.01 g), draw 0.25 mL of 10000 ppm lactoferrin in a 50 mL test tube, add the first phosphate buffer to the scale, mix, centrifuge for 15 min, draw 25 mL of the intermediate layer test solution in another 50 mL centrifuge tube, centrifuge at 5000 r / min for 15 min, draw the supernatant, and wait for purification;

[0194] P1, activation balance: 10mL of the second phosphate buffer

[0195] P2, sample loading: 10 ml of sample solution is loaded;

[0196] P3, first rinse: 10 mL of the second phosphate buffer;

[0197] P4, elution: elution with 4.5 mL of the third phosphate buffer, collecting the eluate, diluting to 5 mL with the third phosphate buffer, mixing, and passing through a hydrophilic PVDF membrane (SCAA-108) for detection;

[0198] P5, Volume adjustment: After nitrogen flushing, use 1mL of acetonitrile-0.1% formic acid solution to adjust the volume to 1mL for injection;

[0199] First phosphate buffer: Weigh 23.85 g of disodium hydrogen phosphate and 4.99 g of sodium dihydrogen phosphate, dissolve in water, adjust the pH to 7.50 with sodium hydroxide solution (4.1), make up to 1 L, and mix thoroughly.

[0200] Second phosphate buffer: Weigh 5.96 g of disodium hydrogen phosphate, 0.96 g of sodium dihydrogen phosphate, and 5.84 g of sodium chloride, dissolve in appropriate amount of water, adjust the pH to 7.50 with sodium hydroxide solution (4.1), make up to 1 L, and mix thoroughly;

[0201] Third phosphate buffer: weigh 5.96 g of disodium hydrogen phosphate, 2.50 g of sodium dihydrogen phosphate, and 119.30 g of sodium chloride, dissolve in appropriate amount of water, adjust the pH to 7.50 with sodium hydroxide solution (4.1), make up to 1 L, and mix thoroughly;

[0202] Sodium hydroxide solution: Weigh 400.00 g of sodium hydroxide, dissolve in water, and dilute to 1 L;

[0203] The detection concentration on the machine was 100 ppm, and the detection instrument was HPLC / UV detection (Waters instrument); the instrument test method referred to the group standard (T / TDSTIA 006-2019): Liquid phase conditions: Chromatographic column: Athena C44.6mm×150mm 5μm; Flow rate: 1mL / min; Wavelength: 280nm; Injection volume: 30μL; Mobile phase: A: 0.1% trifluoroacetic acid, B: acetonitrile; The liquid chromatogram of the standard is shown in Figure 3, the liquid chromatogram corresponding to Example 3 is shown in Figure 4, and the results are shown in Table 1.

[0204] Table 1

[0205] Obviously, Example 4 omits sodium lauryl sulfate, and the amount of dispersant used is less than that of Example 3, which reduces the protection ability of the system, loosens the particles, increases the porosity, and increases the particle size, resulting in an increase in the column flow rate and a decrease in the ion exchange capacity. In Comparative Example 1, after omitting the secondary transfer grafting solution, the grafting amount of heparin functional groups decreases, the corresponding ion exchange capacity decreases, and the recovery rate is poor when high-concentration lactoferrin is spiked. If heparin is directly used as a solid phase extraction column filler, it not only requires a harsh storage environment, but is also prone to clogging the sieve plate or The formation of lumps cannot meet the experimental requirements; Comparative Example 2 only uses the microsphere body to recover lactoferrin, does not contain the functional group of heparin, and only relies on the matrix microspheres bonded with carboxyl functional groups to adsorb the target, which greatly reduces the selectivity of the target lactoferrin. At this time, the matrix microspheres will also adsorb phospholipids or small molecule impurities in matrices such as milk powder, which will interfere with the detection data after sampling; Compared with Example 3, it can be seen that the carboxyl functional group and heparin have a synergistic effect on the target, and can also greatly improve the selectivity of the filler to the target; Comparative Example 3 The surface of the prepared microsphere body does not contain carboxyl functional groups. Since the matrix microspheres are not bonded with carboxyl groups, they cannot form covalent grafts with heparin. The prepared filler mainly interacts with the hydrogen bonding force between the hydroxyl groups on the heparin and the microspheres and the polar forces between the groups. During the pretreatment process, heparin molecules are easily detached from the matrix microspheres, the recovery rate is extremely low, and the interaction between the filler and the target is almost non-existent, so the overall flow rate is fast; Comparative Example 4 has a small particle size, and the matrix is ​​a highly cross-linked spherical agarose, which is in a gel state and requires external pressure to be made The sample liquid passes through the column, and the filler is easy to clog the sieve plate, which is time-consuming and labor-intensive. The particle size of Comparative Example 5 is larger than that of Comparative Example 4, and the uniformity is better, so the column flow rate is slightly faster, but it is still much slower than that of the embodiment, and the sieve plate is easy to clog during the loading process. Due to the low specific surface area, the adsorption capacity for high-concentration target lactoferrin is weak, and its application in actual sample detection is limited. On the other hand, Examples 1-4 have high ion exchange capacity, high particle size, high specific surface area, high lactoferrin recovery rate, and not low column flow rate, can be widely used, and save time and cost.

[0206] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A solid phase extraction column filler, characterized in that: include: A microsphere body formed by copolymerization of a plurality of reactive monomers and a plurality of cross-linking monomers, and a plurality of grafting monomers grafted on the surface of the microsphere body; wherein the reactive monomers are selected from: acrylic acid; The cross-linking monomer is selected from at least one of divinylbenzene, diethylene glycol diacrylate, triethylene glycol diacrylate, or trimethylolpropane trimethacrylate; The grafted monomer is selected from: heparin.

2. A method for preparing a solid phase extraction column filler, characterized in that the steps include: S0-0, providing a reaction container; S0-1. Providing a dispersion solution, wherein the dispersion solution comprises: deionized water and a dispersant dissolved in the deionized water; S0-2, providing a copolymerization solution, wherein the copolymerization solution comprises: a first initiator, a first porogen, a reactive monomer, and a cross-linking monomer; S0-3, providing a grafting solution, wherein the grafting solution comprises: a second initiator, a second porogen, and a grafting monomer; S1, transferring the dispersed solution to the reaction container, transferring the copolymerization solution to the reaction container under stirring conditions, stirring for a first time, gradually heating to a first temperature, and copolymerizing for a second time to obtain a microsphere body; S2, transferring a portion of the grafting solution to the reaction container, and uniformly transferring the catalyst to the reaction container, after the grafting reaction for a third time, gradually heating to a second temperature, transferring another portion of the grafting solution to the reaction container, and grafting reaction for a fourth time, to obtain the microsphere body grafted with the grafting monomer; Wherein, the reactive monomer is selected from: acrylic acid; Wherein, the cross-linking monomer is selected from: divinylbenzene, diethylene glycol diacrylate, triethylene glycol diacrylate, trimethylolpropane trimethacrylate; Wherein, the grafted monomer is selected from: heparin.

3. The preparation method according to claim 2, characterized in that: The dispersant is selected from at least one of polyvinyl pyrrolidone, sodium lauryl sulfate, hydroxypropyl methylcellulose, sodium lignin sulfonate, polyphosphate, or polycarboxylate.

4. The preparation method according to claim 2, characterized in that: The first initiator and the second initiator are independently selected from at least one of benzoyl peroxide, azobisisobutyronitrile, azobisdimethylvaleronitrile, or tert-butylhydroxyperoxide.

5. The preparation method according to claim 2, characterized in that: The first porogen and the second porogen are independently selected from at least one of mesitylene, toluene, n-heptane, cyclohexanol, polystyrene, and polyacrylate.

6. The preparation method according to claim 2, characterized in that: The catalyst is selected from: 4-(4,6-dioxo-1,3,5-triazine-2-yl)-4-methyl-2-oxazolyl-5-oxo-2-chlorophenyl-trifluoroacetic anhydride.

7. The preparation method according to claim 2, characterized in that: The steps also include: S3, spray-drying the microsphere body grafted with the grafted monomer to obtain the solid phase extraction column filler.

8. Application of a solid phase extraction column filler in detecting lactoferrin, characterized in that: The solid phase extraction column filler is as described in claim 1, or the solid phase extraction column filler is prepared by the preparation method according to any one of claims 2-7.

9. A solid phase extraction column, characterized in that: include: The solid phase extraction column filler according to claim 1, or the solid phase extraction column filler obtained by the preparation method according to any one of claims 2 to 7.

10. Application of a solid phase extraction column in detecting lactoferrin, characterized in that: The solid phase extraction column is as described in claim 9.

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