CN108456280B

CN108456280B - Phospholipid organic polymer integral material and preparation method and application thereof

Info

Publication number: CN108456280B
Application number: CN201810051510.XA
Authority: CN
Inventors: 江正瑾; 王启钦; 金含颖; 夏东海; 邵慧凯; 王祥宇
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-01-19
Filing date: 2018-01-19
Publication date: 2020-10-27
Anticipated expiration: 2038-01-19
Also published as: CN108456280A

Abstract

The invention belongs to the technical field of protein purification, and discloses a phospholipid organic polymer integral material, and a preparation method and application thereof. Mixing a phospholipid monomer compound, a cross-linking agent, a pore-forming agent and an initiator, ultrasonically dissolving, degassing, filling into a container, and carrying out polymerization reaction at the temperature of 40-70 ℃ or under the condition of ultraviolet illumination to obtain the phospholipid organic polymer integral material. The material has the advantages of high reproducibility, good biocompatibility and protein nonspecific adsorption resistance, and effectively overcomes the defects of the traditional CRP purification material. The phospholipid bulk material is used as an adsorbent to purify a standard protein mixed sample or an actual sample, the high-purity CRP protein can be obtained by only one-step purification strategy, the operation is simple, and the recovery rate is close to 100%.

Description

Phospholipid organic polymer integral material and preparation method and application thereof

Technical Field

The invention belongs to the technical field of protein purification, and particularly relates to a phospholipid organic polymer integral material, and a preparation method and application thereof.

Background

In 1930 Tillett and Francis found that a substance which reacts with the phospholipid components of the cell wall of streptococcus pneumoniae C-polysaccharide (CPS) to form a precipitate is present in patients with pneumonia [ w.s.tillett, t.jr.francis, j.exp.med.,1930,52:561], hereinafter referred to as C-reactive protein (CRP). Native CRP is a pentameric structure composed of 5 independent subunits, non-covalently, produced by hepatocytes, present mainly in serum, ascites and cerebrospinal fluid [ j.p. atkinson, artritis rheum, 2001,44: 995-; osmand, N.Ertel, J.Siegel, H.Gewurz.Fed.Proc.1975,34:854 ]. In addition, CRP can also be used as an important index for predicting the occurrence of cardiovascular diseases and is closely related to other metabolic syndrome diseases (such as tumors, obesity, intestinal diseases and the like). Subsequent intensive studies find that CRP existing in the body can activate complement system and inhibit the aggregation of specific lymphocytes and platelets, thereby eliminating pathogenic microorganisms invading the body and damaged, necrotic or apoptotic tissue cells, and plays an important role in the natural immune process of the body [ Market B. Pepys, Gideon M. Hischfield, Glenys A. Tennent, et. Nature.2006,440: 1217-. The importance of CRP in organisms has led to the development of more and more extensive studies on its physiological functions, and thus, higher demands have been made on the purity and amount of CRP by physiologists [ John. Volanakis, W.Lowell clements, Ralphe.Schrohenloher, J Immunol Methods,1978,23:285-295 ].

The current methods for purifying CRP are mainly thin-film molecular blotting [ Pei-Chen Chou, John Rick, Tse-Chuan Chou, Anal Chim Acta,2005,542:20-25.]And with Ca²⁺Affinity chromatography with phospholipid-dependent materials as ligands [ L.Soler, N.Garciria, A.Unzueta et al.vet Immunol Immunop,2016,179: 26-31]. The membrane molecular imprinting method can successfully extract CRP in a standard protein mixture, but has poor specificity and can not be repeatedly used, and the practical application value is low. Ca²⁺Phospholipid-dependent affinity chromatography based on Ca²⁺CRP reacts with phospholipid functional groups in the presence of a catalyst to form a precipitateThe method for separation and analysis has the advantages of high purification efficiency and high repeated utilization rate in the purification of CRP, and is widely applied to the enrichment of CRP in body fluids such as ascites, serum and the like.

There are common CRP purification methods [ m.b. pepys, a.c. dash, m.j.ashley.clin.exp.immunol.1977,30,32-37 ] in which Phosphatidylcholine (PC) and Phosphatidylethanolamine (PE) are immobilized as ligands on an agarose carrier, but these methods are complicated in steps, low in recovery rate, and unsatisfactory in one-step purification effect. In particular, agarose is used as a carrier, which may adsorb serum amyloid P component (SAP) having a structure close to that of CRP, and thus, it is necessary to further perform a refining step such as gel filtration or gradient elution to obtain CRP having high purity. Hiroshi Endo et al developed an affinity precipitation method for heat sensitive polymers to purify CRP from rabbit serum in response to the above disadvantages. The method comprises the steps of carrying out polymerization reaction on P-aminophenylphosphorylcholine gel (APPC) and N-isopropylacrylamide (NIPAAm) derivatives to obtain a thermosensitive APPC-polymer, mixing the thermosensitive APPC-polymer with rabbit serum at the critical temperature of below 32 ℃, raising the temperature to above 32 ℃ to form CRP-APPC polymer precipitate, and finally carrying out centrifugal separation to achieve the purpose of enriching and purifying CRP. The CRP obtained by the method has high purity, no SAP pollutants are found, the recovery rate reaches 82.3 percent, and the defect of non-specific adsorption of an agarose carrier is effectively overcome; however, the incubation time of the CRP solution and the thermosensitive polymer in the method is as long as 12 hours and is not repeated, and the subsequent popularization is to be proved [ Mori, S., Nakata, Y., Endo, H., Protein Expres Purif,1994,5: 153-. In recent years, based on the advantages of large specific surface area, good dispersion performance, strong non-specific adsorption resistance and the like of magnetic nano materials, the magnetic nano materials are widely applied to separation and analysis of biological samples [ Horak, D, Babic, M.J.Sep.Sci.,2007,11: 1751-. Eunjoo Kim et al modified 3- (4) -vinylbenzyl-12-phosphocholine dodecanoate (VPC) on magnetic beads to construct VPC-MNPs as an adsorbent to isolate CRP in human serum. The method can achieve the effect of purifying CRP in human serum to a certain extent, but other heteroprotein bands still exist in the purified solution, and the purity is to be further verified; moreover, the method has no investigation on specificity, reproducibility, etc., and is not sufficient for subsequent popularization and use [ Eunjoo Kim, Se Geun Lee, Hyun-ChulKim et al separation Science and Technology,2013,48: 2600-. Thus, currently commercialized CRP purification technology still uses agarose Gel as a carrier and phospholipid material as a ligand (Immobilized p-Aminophenyl phospholipid Choline Gel), but the drawback that specificity and recovery rate cannot be obtained simultaneously is also revealed in subsequent applications [ l.soler, n.garci ia, a.unrueta et al.vet immunological Immunol, 2016,179: 26-31; michael G.Roper, Megan L.Frisk, Janen P.Oberland. anal Chim Acta,2006,569: 195-202 ].

The organic polymer integral material is a polymer formed by free radical polymerization of polymerization liquid of a functional monomer, a cross-linking agent, an initiator and a pore-foaming agent under the condition of light or heat initiation, has simple and rapid preparation process, uniform distribution of large and small pores, good permeability, high stability, good mass transfer rate and excellent biocompatibility, can select a proper functional monomer and a cross-linking agent according to different separation requirements, and has good application prospects in the fields of catalysis, protein purification, separation analysis and the like. Therefore, the novel CRP purification method has the advantages of strong specificity, high recovery rate, good reproducibility, high repeated utilization rate, simple operation steps and capability of effectively avoiding SAP interference, and has great theoretical and practical values.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the invention aims to provide a preparation method of a phospholipid organic polymer integral material.

Another object of the present invention is to provide a phospholipid organic polymer monolith prepared by the above method.

Another object of the present invention is to provide the use of the above phospholipid organic polymer monoliths for the purification of C-reactive proteins.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a phospholipid organic polymer monolithic material comprises the following preparation steps:

mixing a phospholipid monomer compound, a cross-linking agent, a pore-forming agent and an initiator, ultrasonically dissolving, degassing, filling into a container, and carrying out polymerization reaction at the temperature of 40-70 ℃ or under the condition of ultraviolet illumination to obtain the phospholipid organic polymer integral material;

the phospholipid monomer compound refers to 2- (methacryloyloxy) ethyl- [ N- (2-methacryloyloxy) ethyl ] phosphorylcholine (MMPC) compound having the structure of the following formula 1, single-chain Phosphatidylcholine (PC) compound having the structure of the following formula 2, double-chain phosphatidylcholine (DCPC) compound having the structure of the following formula 3 (refer to the references [ Dieter Verzele, Fre 'De' ricLynen, Mike De Vries, et al. development of the first sphingomyelin biological activity phase for immobilized specific membrane, C. 2012,48, 1162-1164. ]), single-chain Phosphatidylethanolamine (PE) compound having the structure of the following formula 4, double-chain phosphatidylethanolamine (DCPE) compound having the structure of the following formula 5, single-chain Phosphatidic Acid (PA) compound having the structure of the following formula 6, double-chain Phosphatidylethanolamine (PS) compound having the structure of the following formula 7, single-chain Phosphatidylserine (PS) compound having the structure of the following formula 8, at least one of double-chain phosphatidylserine (DCPS) compounds with a structure of formula 9, wherein A is N or O element, and the range of N is 2-18;

the phospholipid monomer compound can be prepared by reference to the literature [ Heejin Kim, Wonmin Choi, Seonju Lee et al Synthesis of biomembrane-micron polymers with a variable phospholipid headgroups. Polymer,2014,55,517-524 ].

Preferably, the cross-linking agent is any one of N, N' -Methylenebisacrylamide (MBA), ethylene glycol dimethacrylate (EDMA), and polyethylene glycol diacrylate (poly (ethylene glycol) diacrylate, PDA), and the mass ratio of the phospholipid monomer compound to the cross-linking agent is preferably (1:4) to (4: 1).

Preferably, the porogen is Isopropanol (IPA), Tetrahydrofuran (THF), dimethyl sulfoxide (DMSO)) Methanol (MeOH), cyclohexanol (ANOL), 1,4-Butanediol (BDO), n-dodecanol (1-dodecanol), water (water, H)₂O), wherein the mass ratio of the two porogens is preferably (1:5) - (5: 1); the mass ratio of the phospholipid monomer compound to the pore-forming agent is (1:6) - (1: 1).

Preferably, the initiator is any one of thermal initiator Azobisisobutyronitrile (AIBN) or photoinitiator benzoin bis methyl ether (2,2' -dimethyl-2-phenylacetophenone, DMPA), or 2,2' -Azobisisobutyronitrile amidine dihydrochloride (2,2' -Azobis (2-methyl propioamine), AIBA), Benzoyl Peroxide (BPO), dilauroyl peroxide (LPO), and the amount of the initiator added is preferably 1% of the amount of the phospholipid monomer compound.

Preferably, the container is a stainless steel tube, a glass tube, a capillary tube, a solid phase extraction column, a pipette tip, a solid phase extraction suction head, magnetic nanoparticles, a thin layer plate, filter paper, a filter membrane or a glass monomer bottle.

Preferably, the time of the polymerization reaction is 30-1440 min.

Preferably, after the polymerization reaction is complete, the porogen, unreacted monomer, crosslinker and oligomers are further removed by washing with methanol.

A phospholipid organic polymer monolithic material is prepared by the method.

The phospholipid organic polymer monolithic material is applied to C-reactive protein purification.

Preferably, the application steps of the phospholipid organic polymer monolithic material in the C-reactive protein purification are as follows: balancing the phospholipid monolithic material with a buffer solution A for a period of time, adding a solution to be purified containing CRP, eluting impurities through the buffer solution A, and eluting the specifically adsorbed CRP with a buffer solution B to obtain purified C-reactive protein; the buffer solution A comprises 10mM of Tris (hydroxymethyl) aminomethane (Tris), 50-400 mM of sodium chloride (NaCl) and 2-10 mM of calcium chloride (CaCl)₂) The pH value is 8.0-8.5; the buffer solution B comprises 10mM of Tris (hydroxymethyl) aminomethane (Tris), 50-400 mM of sodium chloride (NaCl), 2-10 mM of disodium ethylene diamine tetraacetate (EDTA-2Na) and 8.0-8.5 of pH.

Preferably, the equilibration time of the buffer solution A is 0-60 min.

Preferably, the solution to be purified containing CRP is a standard protein solution containing CRP, serum of an inflammation patient or an actual sample such as animal serum.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) novel purification materials. Conventional CRP purification materials typically employ phospholipid compounds in combination with an agarose matrix. The phospholipid organic polymer bulk material is used as the adsorbent, so that the characteristics of specific adsorption of CRP by phospholipid compounds and the advantages of simple bulk material preparation, high mass transfer rate, high surface functional group density, good permeability, high reproducibility, good biocompatibility and protein nonspecific adsorption resistance are fully combined, and the defects of the traditional CRP purification material are effectively overcome.

(2) A novel purification process. The traditional CRP purification method mainly comprises the steps of affinity chromatography, gel filtration and the like, and has the disadvantages of complex operation, low purification efficiency and low recovery rate. The method can obtain the high-purity CRP protein by only one-step purification strategy, and has simple operation and a recovery rate close to 100 percent.

(3) The method is simple to operate, high in reuse rate and beneficial to realizing industrialization. Commercial CRP purification column needs to be incubated with sample for 60min before the next operation, and the final CRP contains hetero protein. The phospholipid organic polymer monolithic material adsorbent obtained by the invention does not need to be incubated when the CRP is enriched, the CRP obtained by purification does not contain other hybrid proteins through MALDI-TOF-MS and SDS-PAGE verification, and the recovery rate of the CRP is basically unchanged after the adsorbent is repeatedly used for 98 times.

Drawings

FIG. 1 is a scanning electron micrograph of the internal morphology of the poly (MPC-co-MBA) organic polymer monolith obtained in example 1.

FIG. 2 is a graph showing the energy spectrum analysis of the poly (MPC-co-MBA) organic polymer monolith obtained in example 1.

FIG. 3 is an SDS-PAGE graph (left) and a recovery result graph (right) of the specific selection of CRP and other proteins for the phospholipid organic polymer monolith of example 1.

FIG. 4 is a graph showing the results of the recovery of CRP obtained when the phospholipid organic polymer monolith of example 1 was used continuously for four weeks (98 times in total).

FIG. 5 is a graph showing the results of purifying poly (MPC-co-MBA) organic polymer monoliths against serum (a) and murine plasma (b) from patients with inflammation.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

Preparing a phospholipid monomer compound 2-Methacryloyloxyethyl Phosphorylcholine (MPC), a cross-linking agent MBA, a pore-forming agent (a mixed system of IPA and THF) and an initiator AIBN into a polymerization reaction mixed solution according to an optimized proportion, filling the solution into a 200 mu L pipette tip after ultrasonic dissolution and degassing, then sealing two ends of the pipette tip, and placing the pipette tip into a water bath at 60 ℃ for reaction for 12 hours; after the reaction, the pipette tip is connected with an injector, and the pore-forming agent, the unreacted monomer, the crosslinking agent and the oligomer are washed off by methanol on a peristaltic pump to obtain the phospholipid organic polymer monolithic column (poly (MPC-co-MBA)). Wherein the mass ratio of the monomer MPC to the pore-forming agent is 25:75, the mass ratio of the monomer MPC to the cross-linking agent MBA is 70:30, the mass ratio of the pore-forming agent IPA to the THF is 60:40, and the mass of the initiator AIBN is 1% of that of the monomer MPC.

The Scanning Electron Micrograph (SEM) and the energy spectrum analysis (EDS) of the internal morphology of the poly (MPC-co-MBA) organic polymer monolithic material obtained in the example are shown in FIG. 1 and FIG. 2, respectively. The results in fig. 1 show that the large pores and the small pores in the whole material are uniformly distributed, the pore diameter of the large pores can reach micron level, and the porous structure ensures good permeability of the whole material and the capability of complete protein molecules to pass through. On the other hand, EDS results (fig. 2) of the monolith showed successful bonding of the P element to the polymer surface, confirming that the MPC was successfully immobilized on the monolith substrate.

Example 2

Preparing a phospholipid monomer compound MPC, a cross-linking agent PDA, a pore-forming agent (a mixed system of DMSO and THF) and an initiator AIBN according to an optimized proportion into a polymerization reaction mixed solution, filling the solution into a 200-microliter pipette tip after ultrasonic dissolution and degassing, then sealing two ends of the pipette tip, and placing the pipette tip into a water bath at 60 ℃ for reaction for 12 hours; and after the reaction is finished, connecting the pipette tip with an injector, and washing off the pore-forming agent, the unreacted monomer, the crosslinking agent and the oligomer by using methanol on a peristaltic pump to obtain the poly (MPC-co-PDA) organic polymer integral material. Wherein the mass ratio of the monomer MPC to the pore-forming agent is 15:85, the mass ratio of the monomer MPC to the cross-linking agent PDA is 80:20, the mass ratio of the DMSO to the THF in the pore-forming agent is 40:60, and the mass of the initiator is 1% of the total mass of the monomer MPC.

Example 3

Preparing a phospholipid monomer compound MPC, a cross-linking agent EDMA, a pore-forming agent (a mixed system of IPA and BDO) and an initiator AIBN into a polymerization reaction mixed solution according to an optimized proportion, filling the solution into a 200-microliter pipette tip after ultrasonic dissolution and degassing, then sealing two ends of the pipette tip, and putting the pipette tip into a water bath at 60 ℃ for reaction for 12 hours; and after the reaction is finished, connecting the pipette tip with an injector, and washing away unreacted monomers, pore-forming agents and oligomers on a peristaltic pump by using methanol to obtain the poly (MPC-co-EDMA) organic polymer integral material. Wherein the mass ratio of the monomer MPC to the pore-forming agent is 15:85, the mass ratio of the monomer MPC to the cross-linking agent EDMA is 53:47, the mass ratio of IPA to BDO in the pore-forming agent is 25:75, and the mass of the initiator is 1% of the mass of the monomer MPC.

Example 4

Preparing a phospholipid monomer compound 2-methacryloyloxydodecyl phosphatidylcholine (MDPC), a cross-linking agent EDMA, a pore-forming agent (a mixed system of IPA and BDO) and an initiator AIBN into a polymerization reaction mixed solution according to an optimized proportion, ultrasonically dissolving and degassing, filling into a 200 mu L pipette tip, sealing two ends of the pipette tip, and placing into a water bath at 60 ℃ for reaction for 12 hours; after the reaction, the pipette tip is connected with an injector, and the pore-forming agent, the unreacted monomer, the cross-linking agent and the oligomer are washed away by methanol on a peristaltic pump to obtain the poly (MDPC-co-EDMA) organic polymer integral material. Wherein the mass ratio of the monomer MDPC to the pore-forming agent is 25:75, the mass ratio of the monomer MDPC to the cross-linking agent EDMA is 40:60, the mass ratio of IPA to BDO in the pore-forming agent is 83.3:16.7, and the mass of the initiator is 1% of the mass of the monomer MDPC.

Example 5

Preparing a phospholipid monomer compound 1-lauroyl-2- (11-methacrylamide undecanoyl) -SN-glycerol-3-phosphatidylcholine (1-dodecanoyl-2- (11-methacrylamido-decanoyl) -SN-glycero-3-phosphatidylcholine, MDSPC), a cross-linking agent EDMA, a pore-forming agent (a mixed system of IPA and ANOL) and an initiator AIBN according to an optimized proportion into a polymerization reaction mixed solution, ultrasonically dissolving, degassing, filling into a 200-microliter pipette tip, sealing two ends of the pipette tip, and placing into a water bath at 60 ℃ for reaction for 12 hours; after the reaction, the pipette tip is connected with an injector, and the pore-forming agent, the unreacted monomer, the cross-linking agent and the oligomer are washed away by methanol on a peristaltic pump to obtain the poly (MDSPC-co-EDMA) organic polymer monolithic material. Wherein the mass ratio of the monomer MDSPC to the pore-forming agent is 27:73, the mass ratio of the monomer MDSPC to the cross-linking agent EDMA is 45:55, the mass ratio of IPA to ANOL in the pore-forming agent is 83.3:16.7, and the mass of the initiator is 1% of that of the monomer MDSPC.

Example 6

Preparing a phospholipid zwitterionic compound MMPC, a pore-forming agent (a mixed system of MeOH and THF) and an initiator AIBN according to an optimized proportion into a polymerization reaction mixed solution, ultrasonically dissolving, degassing, filling into a 200 mu L pipette tip, sealing two ends of the pipette tip, and placing into a water bath at 60 ℃ for reaction for 12 hours; and after the reaction is finished, connecting the pipette tip with an injector, and washing off the pore-forming agent and unreacted monomers and oligomers on a peristaltic pump by using methanol to obtain the poly MMPC organic polymer integral material. Wherein the mass ratio of the MMPC to the pore-foaming agent is 30:70, the mass ratio of MeOH to THF in the pore-foaming agent is 35:65, and the mass of the initiator is 1% of the mass of the MMPC.

The phospholipid organic polymer monolithic columns of the above examples were used for C-reactive protein purification:

firstly, the organic polymer monolithic material obtained in example 1 is used as an adsorbent, and the specific selectivity of the organic polymer monolithic material on CRP is considered:

the specific selectivity of the adsorbent for CRP was examined by using a mixed sample containing Human Serum Albumin (HSA), human immunoglobulin g (igg), beta lactoglobulin (β -lactoglobulin), lysozyme (lysozyme), cytochrome c (cytochrome c), and CRP as a test sample. The test conditions were:

adsorbent: poly (MPC-co-MBA) organic polymer monoliths;

sample preparation: HSA, IgG, beta-lactoglulin, lysozyme, cytochrome C;

leacheate: buffer solution A (composition of 10mM Tris, 140mM NaCl, 2mM CaCl₂)，pH＝8.0～8.5)；

Eluent: buffer solution B (composition of 10mM Tris, 140mM sodium chloride (NaCl), 2mM disodium ethylenediaminetetraacetate (EDTA-2Na), pH 8.0 to 8.5.)

Flow rate: 100 μ L/min.

The obtained SDS-PAGE (left) and recovery rate (right) of the phospholipid organic polymer monolithic material for the specific selection of CRP and other proteins are shown in FIG. 3, and the results in FIG. 3 show that the impure proteins can be completely washed by the buffer solution A in the washing step, and only CRP exists in the eluent, which proves that the adsorbent has very good specific selectivity for CRP.

Secondly, the organic polymer monolithic material obtained in example 1 is used for investigating stability and recycling rate when continuously purifying samples:

the test conditions were:

adsorbent: poly (MPC-co-MBA) organic polymer monoliths;

sample preparation: a standard CRP solution;

leacheate: a buffer solution A;

eluent: a buffer solution B;

flow rate: 100 μ L/min.

The recovery rate of the CRP obtained after the continuous use for four weeks (98 times in total) is shown in FIG. 4, and the results in FIG. 4 show that the recovery rate of the CRP obtained after the continuous use for four weeks (98 times in total) of the poly (MPC-co-MBA) organic polymer monolithic material is not obviously changed, which proves that the adsorbent has good stability and high reuse rate in the whole purification process.

And thirdly, using the organic polymer monolithic material obtained in the example 1 for purifying CRP in actual biological samples:

the purification conditions were:

adsorbent: poly (MPC-co-MBA) organic polymer monoliths;

sample preparation: a: serum from patients with inflammation; b: rat plasma;

leaching the solution: a buffer solution A;

elution solution: a buffer solution B;

total flow rate: 100 mu L/min;

the figure 5 shows that CRP in the sample can be enriched by the adsorbent, and other proteins can not be adsorbed on the monolithic column, thus proving that the phospholipid organic polymer monolithic column prepared by the invention can be successfully applied to the purification of CRP in actual samples.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The application of the phospholipid organic polymer monolithic material in the purification of C-reactive protein is characterized by comprising the following steps: balancing the phospholipid organic polymer monolithic material with a buffer solution A for a period of time, adding a solution to be purified containing CRP, eluting impurities through the buffer solution A, and eluting the specifically adsorbed CRP with a buffer solution B to obtain purified C-reactive protein; the buffer solution A comprises 10mM of trihydroxymethyl aminomethane, 50-400 mM of sodium chloride and 2-10 mM of calcium chloride, and the pH value is 8.0-8.5; the buffer solution B comprises 10mM of trihydroxymethyl aminomethane, 50-400 mM of sodium chloride and 2-10 mM of disodium ethylene diamine tetraacetate, and the pH value is 8.0-8.5;

the phospholipid organic polymer monolithic material is prepared by the following method:

the phospholipid monomer compound is at least one of a 2- (methacryloyloxy) ethyl- [ N- (2-methacryloyloxy) ethyl ] phosphorylcholine compound with a structure shown in a formula 1, a single-chain phosphatidylcholine compound with a structure shown in a formula 2, a double-chain phosphatidylcholine compound with a structure shown in a formula 3, a single-chain phosphatidylethanolamine compound with a structure shown in a formula 4, a double-chain phosphatidylethanolamine compound with a structure shown in a formula 5, a single-chain phosphatidic acid compound with a structure shown in a formula 6, a double-chain phosphatidic acid compound with a structure shown in a formula 7, a single-chain phosphatidylserine compound with a structure shown in a formula 8 and a double-chain phosphatidylserine compound with a structure shown in a formula 9, wherein A is N or O element, and N ranges from 2 to 18;

2. the use of the phospholipid organic polymer monolithic material of claim 1, wherein the phospholipid organic polymer monolithic material comprises: the cross-linking agent is any one of N, N' -methylene bisacrylamide, ethylene glycol dimethacrylate and polyethylene glycol diacrylate, and the mass ratio of the phospholipid monomer compound to the cross-linking agent is (1:4) - (4: 1).

3. The use of the phospholipid organic polymer monolithic material of claim 1, wherein the phospholipid organic polymer monolithic material comprises: the pore-foaming agent is a binary mixed system consisting of any two of isopropanol, tetrahydrofuran, dimethyl sulfoxide, methanol, cyclohexanol, 1,4-butanediol, n-dodecanol and water, wherein the mass ratio of the two pore-foaming agents is (1:5) - (5: 1); the mass ratio of the phospholipid monomer compound to the pore-forming agent is (1:6) - (1: 1).

4. The use of the phospholipid organic polymer monolithic material of claim 1, wherein the phospholipid organic polymer monolithic material comprises: the initiator is thermal initiator azobisisobutyronitrile or photoinitiator benzoin dimethyl ether, or any one of 2,2' -azobisisobutyronitrile amidine dihydrochloride, benzoyl peroxide and dilauryl peroxide, and the addition amount of the initiator is 1% of the mass of the phospholipid monomer compound.

5. The use of the phospholipid organic polymer monolithic material of claim 1, wherein the phospholipid organic polymer monolithic material comprises: the container is a stainless steel tube, a glass tube, a capillary tube, a solid phase extraction column, a pipette tip, a solid phase extraction suction head, magnetic nanoparticles, a thin layer plate, filter paper, a filter membrane or a glass monomer bottle.

6. The use of the phospholipid organic polymer monolithic material of claim 1, wherein the phospholipid organic polymer monolithic material comprises: the polymerization reaction time is 30-1440 min; after the polymerization was complete, the porogen, unreacted monomer, crosslinker and oligomers were further removed by washing with methanol.

7. The use of the phospholipid organic polymer monolithic material of claim 1, wherein the phospholipid organic polymer monolithic material comprises: the balance time of the buffer solution A is 0-60 min; the solution to be purified containing CRP is standard protein solution containing CRP, serum of inflammation patients or animal serum.