CN108456280A - A kind of phospholipid organic polymer integral material and its preparation method and application - Google Patents
A kind of phospholipid organic polymer integral material and its preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
- C08F230/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4737—C-reactive protein
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F130/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
- C08F130/02—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2343/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing boron, silicon, phosphorus, selenium, tellurium or a metal; Derivatives of such polymers
- C08J2343/02—Homopolymers or copolymers of monomers containing phosphorus
Abstract
The invention belongs to Protein purification techniques field, a kind of phospholipid organic polymer integral material and its preparation method and application is disclosed.Phospholipid monomeric compound, crosslinking agent, pore-foaming agent and initiator are mixed, then poured into container after ultrasonic dissolution, degassing, the polymerisation at 40~70 DEG C of temperature or ultraviolet irradiation condition obtains the phospholipid organic polymer integral material.The material has the advantage of reproducibility height, good biocompatibility and anti-albumen non-specific adsorption, effectively overcomes the defect present in traditional CRP purified materials.Using the phospholipid integral material of the present invention as adsorbent purification standard albumen mixing sample or actual sample, it is only necessary to can be obtained the CRP albumen of high-purity by a step purification strategy, and the easy to operate, rate of recovery is close to 100%.
Description
Technical field
The invention belongs to Protein purification techniques fields, and in particular to a kind of phospholipid organic polymer integral material and its system
Preparation Method and application.
Background technology
Nineteen thirty, Tillett and Francis have found that there are a kind of energy and streptococcus pneumonia C polysaccharide in patients with pneumonia body
(CPS) the phosphatide constituents on cell wall react generate precipitation substance [W.S.Tillett, T.Jr.Francis,
J.Exp.Med.,1930,52:561], referred to as C reactive protein (CRP) afterwards.Natural CRP is one kind by 5 independent subunits
The pentamer structure of non-covalent composition, is generated by liver cell, is primarily present in serum, ascites and cerebrospinal fluid
[J.P.Atkinson,Arthritis Rheum.,2001,44:995-996.], content is very low in normal body, inflammation
The increase that content can be hundreds and thousands of times when reaction, therefore become a kind of nonspecific acute phase protein
[M.McCarty,J.Exp.Meal.,1947,85:491.；A.P.Osmand,N.Ertel,J.Siegel,
H.Gewurz.Fed.Proc.1975,34:854.].In addition, CRP can also be used as the important finger that prediction angiocardiopathy occurs
Mark, and (such as with other metabolic syndrome diseases：Tumour, obesity, enteron aisle disease etc.) it is in close relations.Follow-up further investigation
It was found that the CRP being present in body can also activating complement system, inhibit the aggregation of specific lymphocytes and platelets, to clear
Except the pathogenic microorganism and damage of invasion body, the histocyte of necrosis or apoptosis, played during the body innate immunity
Important role [Market B.Pepys, Gideon M.Hischfield, Glenys A.Tennent, et
al.Nature.2006,440:1217-1220.].The importance of CRP in vivo makes the research of its physiologic function more next
It is more extensive, therefore purity and dosage of the physiologist to CRP more stringent requirements are proposed [Johne.Volanakis, W.Lowell
Clements,Ralphe.Schrohenloher,J Immunol Methods,1978,23:285-295.]。
The method of purifying CRP mainly has membrane molecular blotting [Pei-Chen Chou, John Rick, Tse- at present
Chuan Chou,Anal Chim Acta,2005,542:20-25.] and with Ca2+The phospholipid material of dependence is as aglucon
Affinity chromatography [L.Soler, N.Garc í a, A.Unzueta et al.Vet Immunol Immunop, 2016,179:26–
31].Membrane molecular blotting can successfully extract the CRP in standard protein mixture, but its specificity is bad and can not repeat to make
With actual application value is little.Ca2+The phospholipid affinity chromatography of dependence is based in Ca2+Under existence condition CRP can and
Phospholipid functional group reactions simultaneously generate a kind of method for precipitating and then carrying out separation analysis, are presented in the purifying of CRP pure
Change that efficiency is higher, the preferable advantage of recycling rate of waterused, is widely used in the enrichment of CRP in the body fluid such as ascites, serum.
It common are and be fixed on phosphatidyl choline (PC), phosphatidyl-ethanolamine (PE) on agarose carrier as aglucon
CRP purification process [M.B.Pepys, A.C.Dash, M.J.Ashley.Clin.exp.Immunol.1977,30,32-37.], but
It is that these method and steps are cumbersome, the rate of recovery is low and a step purification effect is undesirable.It, can in particular by agarose as carrier
It can adsorb and serum amyloid P component (SAP) similar in CRP structures, it is therefore desirable to further pass through gel filtration or ladder
The purification steps such as degree elution could obtain the CRP of high-purity.Hiroshi Endo etc. are directed to disadvantages mentioned above, develop a kind of temperature-sensitive
Property polymer affinity precipitation purifying rabbit anteserum in CRP.They pass through P- aminobenzene Phosphorylcholine gels (APPC) and N-
The derivative of N-isopropylacrylamide (NIPAAm) occurs polymerisation and obtains thermal sensitivity APPC- polymer, and by it critical
After 32 DEG C of temperature or less is mixed with rabbit anteserum, temperature rise is to 32 DEG C or more and then forms CRP-APPC polymer precipitation, finally
Achieve the purpose that enriching and purifying CRP by centrifuging.CRP purity obtained by this method is high and without finding SAP pollutants, recycling
Rate reaches 82.3%, effectively compensates for the defect of agarose carrier non-specific adsorption；But CRP solution and temperature-sensitive in this method
Property polymer incubation time up to 12h and not reappearing, it is follow-up promote need to be proved [Mori, S., Nakata, Y., Endo,
H.,Protein Expres Purif,1994,5:153-156.].In recent years, it is based on magnetic Nano material large specific surface area, point
Dissipate the advantages that performance is good, and anti-non-specific adsorption performance is strong, be widely used to biological sample separation analysis [Horak, D.,
Babic,M.,Mackova,H.,Benes,M.J.Sep.Sci.,2007,11:1751-1772.].Eunjoo Kim etc. are by 3-
(4) in-vinyl benzyl -12- phosphocholines dodecylate (VPC) modification to magnetic bead, structure VPC-MNPs carriers are as suction
CRP in attached dose of separation human serum.This method can achieve the effect that CRP in step purifying human serum to a certain extent, still
Still there are the presence of other foreign protein bands, purity to need further to be verified in solution after purification；And this method does not have
The investigations such as specificity, reproducibility are carried out, are not enough to subsequently promote the use of [Eunjoo Kim, Se Geun Lee, Hyun-Chul
Kim et al.Separation Science and Technology,2013,48:2600-2607].Therefore commercialization at present
CRP purification techniques still use Ago-Gel as carrier, phospholipid material is as aglucon (Immobilized p-
Aminophenyl Phosphoryl Choline Gel), but its specific the drawbacks of can not getting both with rate of recovery, is also follow-up
Shown in [L.Soler, N.Garc í a, A.Unzueta et al.Vet Immunol Immunop, 2016,179:
26–31；Michael G.Roper,Megan L.Frisk,Janeen P.Oberlander.Anal Chim Acta,2006,
569:195–202]。
Organic polymer integral material is that the polymer fluid of function monomer, crosslinking agent, initiator and pore-foaming agent draws in light or heat
Clockwork spring part issues raw Raolical polymerizable and the polymer that is formed, preparation process is simple and quick, size pore size distribution uniformly, ooze
Permeability is good, stability is high and has good mass transfer rate and excellent biocompatibility, and can be according to different separation demands
Suitable function monomer and crosslinking agent are selected, has been shown well in fields such as catalysis, protein purification and separation analyses
Application prospect.Therefore exploitation high specificity, rate of recovery height, favorable reproducibility, high recycling rate, operating procedure are simple and can be effective
The CRP Novel purification method new materials for avoiding SAP from interfering, theoretical and real value are very great.
Invention content
In place of the above shortcoming and defect of the existing technology, the primary purpose of the present invention is that providing a kind of phosphatide
The preparation method of class organic polymer integral material.
Another object of the present invention is to provide a kind of phospholipid organic polymer being prepared by the above method is whole
Material.
It is still another object of the present invention to provide above-mentioned phospholipid organic polymer integral materials in purifying C reactive protein
In application.
The object of the invention is achieved through the following technical solutions：
A kind of preparation method of phospholipid organic polymer integral material, including following preparation process：
Phospholipid monomeric compound, crosslinking agent, pore-foaming agent and initiator are mixed, then poured into after ultrasonic dissolution, degassing
In container, the polymerisation at 40~70 DEG C of temperature or ultraviolet irradiation condition obtains the phospholipid organic polymer entirety material
Material；
The phospholipid monomeric compound refers to 2- (methacryloxy) ethyls-[N- such as 1 structure of following formula
(2- methacryloxies) ethyl] Phosphorylcholine (MMPC) compound, single-stranded phosphatidyl choline (PC) chemical combination of 2 structure of formula
Dichain phospholipids phatidylcholine (DCPC) compound of object, 3 structure of formula (can refer to document [Dieter Verzele, Fre ' de ' ric
Lynen,Mike De Vrieze,et al.Development of the first sphingomyelin biomimetic
stationary phase for immobilized artificial membrane(IAM)
Chromatography.Chem.Commun., 2012,48,1162-1164.]), the single-stranded phosphatidyl-ethanolamine of 4 structure of formula
(PE) compound, dichain phospholipids acyl ethanol amine (DCPE) compound of 5 structure of formula, single-stranded phosphatidic acid (PA) chemical combination of 6 structure of formula
Object, sour (DCPA) compound of dichain phospholipids of 7 structure of formula, single-stranded phosphatidylserine (PS) compound of 8 structure of formula, 9 knot of formula
At least one of dichain phospholipids acyl serine (DCPS) compound of structure, A is N or O elements in formula, and the range of n is 2~18；
Above-mentioned phospholipid monomeric compound can refer to document [Heejin Kim, Wonmin Choi, Seonju Lee et
al.Synthesis of biomembrane-mimic polymers with various phospholipid head
Groups.Polymer, 2014,55,517-524.] it is prepared.
Preferably, the crosslinking agent is N, N'- methylene-bisacrylamides (N, N'-Methylenebisacrylamid
E, MBA), ethylene glycol dimethacrylate (Ethyleneglycol dimethacrylate, EDMA), polyethylene glycol dipropyl
Any one in olefin(e) acid ester (Poly (ethylene glycol) diacrylate, PDA), the phospholipid monomeric compound
Mass ratio with crosslinking agent is preferably (1:4)~(4:1).
Preferably, the pore-foaming agent be isopropanol (isopropanol, IPA), tetrahydrofuran (tetrahydrofuran,
THF), dimethyl sulfoxide (DMSO) (dimethyl sulfoxide, DMSO), methanol (methanol, MeOH), cyclohexanol
(cyclohexanol, ANOL), Isosorbide-5-Nitrae butanediol (Isosorbide-5-Nitrae-butanediol, BDO), n-dodecanol (1-dodecanol,
LOROL), water (water, H2O the arbitrary two kinds double base mixed systems formed in), the mass ratio of two of which pore-foaming agent are excellent
It is selected as (1:5)~(5:1)；The mass ratio of the phospholipid monomeric compound and pore-foaming agent is (1:6)~(1:1).
Preferably, the initiator be thermal initiator azodiisobutyronitrile (Azobisisobutyronitrile,
AIBN) or photoinitiator benzoin dimethylether (2,2 '-Dimethoxy-2-phenylacetophenone, DMPA) or 2,2 '-
Azodiisobutyronitrile amidine dihydrochloride (2,2'-Azobis (2-methylpropionamidine) dihydrochloride,
AIBA), benzoyl peroxide (Dibenzoyl peroxide, BPO), the double lauroyl of peroxidating (Lauroyl peroxide,
LPO any one in), the addition of the initiator is preferably the 1% of phospholipid monomeric compound quality.
Preferably, the container is stainless steel tube, glass tube, capillary, solid phase extraction column, liquid transfer gun head, solid phase
Extract suction nozzle, magnetic nanoparticle, lamellae, filter paper, filter membrane or glass monolith bottle.
Preferably, the time of the polymerisation is 30~1440min.
Preferably, it after the completion of the polymerisation, is further rinsed with methanol and removes pore-foaming agent, unreacted monomer, friendship
Join agent and oligomer.
A kind of phospholipid organic polymer integral material, is prepared by the above method.
Application of the above-mentioned phospholipid organic polymer integral material in C reactive protein purifying.
Preferably, applying step of the phospholipid organic polymer integral material in C reactive protein purifying is as follows：
Phospholipid integral material is first used to buffer solution A balance a period of times, the solution to be purified containing CRP is added, then by slow
Solution A elution impurity is rushed, finally uses buffer solution B elutions by the CRP of specific adsorption, obtains C- reaction eggs after purification
In vain；The group of the buffer solution A become 10mM trishydroxymethylaminomethanes (Tris), 50~400mM sodium chloride (NaCl), 2~
10mM calcium chloride (CaCl2), pH=8.0~8.5；The group of the buffer solution B becomes 10mM trishydroxymethylaminomethanes
(Tris), 50~400mM sodium chloride (NaCl), 2~10mM disodium ethylene diamine tetraacetate (EDTA-2Na), pH=8.0~8.5.
Preferably, the buffer solution A equilibration times are 0~60min.
Preferably, the solution to be purified containing CRP be standard protein solution containing CRP, inflammatory patients serum or dynamic
The actual samples such as object serum.
Compared with the existing technology, the invention has the advantages that and advantageous effect：
(1) novel purified material.Traditional CRP purified material generally use phospholipids compounds are mutually tied with agarose matrix
It closes.And the present invention uses phospholipid organic polymer integral material that it is special fully to combine phospholipids compounds as adsorbent
Property absorption CRP characteristic and integral material prepare that simple, mass transfer rate is fast, surface functional group density is big, good penetrability, reproducibility
High, good biocompatibility and anti-albumen non-specific adsorption advantage effectively overcomes lacking present in traditional CRP purified materials
It falls into.
(2) novel purification process.Traditional CRP purification process includes mainly affinity chromatography and gel filtration, behaviour
Make that cumbersome, purification efficiency is low and the rate of recovery is not high.And the method for the invention only needs to can be obtained height by a step purification strategy
The CRP albumen of purity and the easy to operate, rate of recovery are close to 100%.
(3) easy to operate, reuse rate is high, be conducive to realize industrialization.When commercialized CRP purifies small column purification CRP
Need to next step operation could be carried out after sample incubation 60min, and contain foreign protein in the CRP finally purified.And institute of the present invention
Without hatching when obtaining phospholipid organic polymer integral material adsorbent enrichment CRP, the CRP purified passes through MALDI-TOF-
MS and SDS-PAGE verifications are substantially unchanged to the rate of recovery of CRP after being reused 98 times without other foreign proteins, and adsorbent.
Description of the drawings
Fig. 1 is the scanning of poly (MPC-co-MBA) the organic polymer integral material internal morphology obtained in embodiment 1
Electron microscope.
Fig. 2 is the energy spectrum analysis figure of poly (MPC-co-MBA) the organic polymer integral material obtained in embodiment 1.
Fig. 3 is specific selection of the phospholipid organic polymer integral material to CRP and other albumen of embodiment 1
SDS-PAGE schemes (left side) and rate of recovery result figure (right side).
Fig. 4 is gained after surrounding (amounting to 98 times) is used continuously in the phospholipid organic polymer integral material of embodiment 1
The rate of recovery result figure of CRP.
Fig. 5 is poly (MPC-co-MBA) organic polymer integral materials to inflammatory patients serum (a) and mouse blood plasma (b)
Purification result figure.
Specific implementation mode
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
By phospholipid monomeric compound 2- methylacryoyloxyethyl phosphocholines (2-methacryloyloxyethyl
Phosphorylcholine, MPC), crosslinking agent MBA, pore-foaming agent (mixed system of IPA and THF) and initiator A IBN, according to
Optimization ratio is configured to polymerisation mixed solution, is poured into after ultrasonic dissolution, degassing in 200 μ L liquid transfer gun heads, then by liquid relief
Pipette tips sealing two ends are put into 60 DEG C of water-baths and react 12h；Liquid transfer gun head is connect with syringe after completion of the reaction, in peristaltic pump
On rinse out pore-foaming agent, unreacted monomer, crosslinking agent and oligomer with methanol and obtain phospholipid Organic Polymer Monolithic Columns
(poly(MPC-co-MBA)).Wherein the mass ratio of monomer MPC and pore-foaming agent is 25:75, the matter of monomer MPC and crosslinking agent MBA
Amount is than being 70:30, the mass ratio of pore-foaming agent IPA and THF are 60:The quality of 40, initiator A IBN are the 1% of monomer MPC.
The scanning electron microscope of poly (MPC-co-MBA) the organic polymer integral material internal morphology obtained in the present embodiment
Scheme (SEM) and energy spectrum analysis figure (EDS) is as depicted in figs. 1 and 2 respectively.The integral material interior size hole point known to Fig. 1 results
Cloth is uniformly and macropore diameter can reach micron order, and this porous structure ensure that the good permeability of integral material and complete egg
White molecule by ability.On the other hand, the EDS results (Fig. 2) of integral material show that P element is successfully bonded to polymer table
Face, it was confirmed that MPC is successfully fixed in integral material matrix.
Embodiment 2
By phospholipid monomeric compound MPC, crosslinking agent PDA, pore-foaming agent (mixed system of DMSO and THF) and initiator
AIBN is configured to polymerisation mixed solution according to optimization ratio, is poured into after ultrasonic dissolution, degassing in 200 μ L liquid transfer gun heads, so
Afterwards by liquid transfer gun head sealing two ends, it is put into 60 DEG C of water-baths and reacts 12 hours；Liquid transfer gun head and syringe are connected after completion of the reaction
It connects, rinses out pore-foaming agent and unreacted monomer, crosslinking agent and oligomer with methanol on peristaltic pump, obtain poly (MPC-co-
PDA) organic polymer integral material.Wherein the mass ratio of monomer MPC and pore-foaming agent is 15:85, monomer MPC and crosslinking agent PDA
Mass ratio be 80:20, the mass ratio of DMSO and THF is 40 in pore-foaming agent:60, the quality of initiator is monomer MPC total amounts
1%.
Embodiment 3
By phospholipid monomeric compound MPC, crosslinking agent EDMA, pore-foaming agent (mixed system of IPA and BDO) and initiator
AIBN is configured to polymerisation mixed solution according to optimization ratio, is poured into after ultrasonic dissolution, degassing in 200 μ L liquid transfer gun heads, so
Afterwards by liquid transfer gun head sealing two ends, it is put into 60 DEG C of water-baths and reacts 12h；Liquid transfer gun head is connect with syringe after completion of the reaction,
Unreacted monomer, pore-foaming agent and oligomer are rinsed out with methanol on peristaltic pump, it is organic poly- to obtain poly (MPC-co-EDMA)
Close object integral material.Wherein the mass ratio of monomer MPC and pore-foaming agent is 15:85, monomer MPC and the mass ratio of crosslinking agent EDMA are
53:47, the mass ratio of IPA and BDO is 25 in pore-foaming agent:75, the quality of initiator is the 1% of monomer MPC mass.
Embodiment 4
By phospholipid monomeric compound 2- methacryloxypropyl dodecyl phosphatidyl cholines (12-methacryloyl
Dodecylphosphocholine, MDPC), crosslinking agent EDMA, pore-foaming agent (mixed system of IPA and BDO) and initiator
AIBN is configured to polymerisation mixed solution according to optimization ratio, is poured into after ultrasonic dissolution, degassing in 200 μ L liquid transfer gun heads, so
Afterwards by liquid transfer gun head sealing two ends, it is put into 60 DEG C of water-baths and reacts 12 hours；Liquid transfer gun head and syringe are connected after completion of the reaction
It connects, rinses out pore-foaming agent and unreacted monomer, crosslinking agent and oligomer with methanol on peristaltic pump, obtain poly (MDPC-
Co-EDMA) organic polymer integral material.Wherein the mass ratio of monomer MDPC and pore-foaming agent is 25:75, monomer MDPC and crosslinking
The mass ratio of agent EDMA is 40:60, the mass ratio of IPA and BDO is 83.3 in pore-foaming agent:16.7, the quality of initiator is monomer
The 1% of MDPC mass.
Embodiment 5
By phospholipid monomeric compound 1- dodecanoyls -2- (11- Methacrylamides undecanoyl)-SN- glycerine -3- phosphatide
Phatidylcholine (1-dodecanoyl-2- (11-methacrylamidoundecanoyl)-sn-glycero-3-
Phosphocholine, MDSPC), crosslinking agent EDMA, pore-foaming agent (mixed system of IPA and ANOL) and initiator A IBN, according to
Optimization ratio is configured to polymerisation mixed solution, is poured into after ultrasonic dissolution, degassing in 200 μ L liquid transfer gun heads, then by liquid relief
Pipette tips sealing two ends are put into 60 DEG C of water-baths and react 12 hours；Liquid transfer gun head is connect with syringe after completion of the reaction, is being wriggled
Pore-foaming agent and unreacted monomer, crosslinking agent and oligomer are rinsed out with methanol on pump, obtaining poly (MDSPC-co-EDMA) has
Machine polymer integral material.Wherein the mass ratio of monomer MDSPC and pore-foaming agent is 27:73, monomer MDSPC are with crosslinking agent EDMA's
Mass ratio is 45:55, the mass ratio of IPA and ANOL is 83.3 in pore-foaming agent:16.7, the quality of initiator is monomer MDSPC matter
The 1% of amount.
Embodiment 6
By phospholipid zwitterionic compound MMPC, pore-foaming agent (mixed system of MeOH and THF) and initiator A IBN, press
It is configured to polymerisation mixed solution according to optimization ratio, is poured into after ultrasonic dissolution, degassing in 200 μ L liquid transfer gun heads, then will be moved
Liquid pipette tips sealing two ends are put into 60 DEG C of water-baths and react 12 hours；Liquid transfer gun head is connect with syringe after completion of the reaction, compacted
Pore-foaming agent and unreacted monomer and oligomer are rinsed out with methanol on dynamic pump, obtains poly MMPC organic polymer entirety materials
Material.The mass ratio of wherein MMPC and pore-foaming agent is 30:70, the mass ratio of MeOH and THF is 35 in pore-foaming agent:65, initiator
Quality is the 1% of MMPC mass.
Application effect test of the phospholipid Organic Polymer Monolithic Columns of above-described embodiment in C reactive protein purifying：
One, using 1 gained organic polymer integral material of embodiment as adsorbent, its specific selectivity to CRP is investigated：
To contain human serum albumins (HSA), immunoglobulin G (IgG), beta lactoglobulin (β-
Lactoglobulin), lysozyme (lysozyme), cromoci (cytochrome C), CRP sample mixing be test sample,
Investigate specific selectivity of the adsorbent to CRP.Test condition is：
Adsorbent：Poly (MPC-co-MBA) organic polymer integral material；
Sample：HSA、IgG、β-lactoglobulin、lysozyme、cytochrome C；
Leacheate：Buffer solution A (organizing becomes 10mM trishydroxymethylaminomethanes (Tris), 140mM sodium chloride (NaCl),
2mM calcium chloride (CaCl2), pH=8.0~8.5)；
Eluent：(group becomes 10mM trishydroxymethylaminomethanes to buffer solution B as the group of the buffer solution B
(Tris), 140mM sodium chloride (NaCl), 2mM disodium ethylene diamine tetraacetate (EDTA-2Na), pH=8.0~8.5.)
Flow velocity：100μL/min.
SDS-PAGE of the obtained phospholipid organic polymer integral material to the specific selection of CRP and other albumen
(left side) and rate of recovery result figure (right side) are schemed as shown in figure 3, by Fig. 3 results it is found that foreign protein can be buffered in rinsing step
Solution A is rinsed completely, and CRP is only existed in eluent, this demonstrate that the adsorbent has extraordinary special selection to CRP
Property.
Two, with 1 gained organic polymer integral material of embodiment, for the stability and again when investigating continuous purification sample
Multiple utilization rate：
Test condition is：
Adsorbent：Poly (MPC-co-MBA) organic polymer integral material；
Sample：Standard CRP solution；
Leacheate：Buffer solution A；
Eluent：Buffer solution B；
Flow velocity：100μL/min.
After surrounding (amounting to 98 times) is used continuously, the rate of recovery of gained CRP is as shown in figure 4, by Fig. 4 results it is found that poly
(MPC-co-MBA) after organic polymer integral material continuous use surrounding (amounting to 98 times), the rate of recovery of gained CRP is not bright
Aobvious variation, it was demonstrated that the adsorbent has good stability in whole process of purification, and reuse rate is high.
Three, with the organic polymer integral material of 1 gained of embodiment, the purifying for CRP in practical biological sample：
Purification condition is：
Adsorbent：Poly (MPC-co-MBA) organic polymer integral material；
Sample：a：Inflammatory patients serum；b：Mouse blood plasma；
Eluent solution：Buffer solution A；
Elute solution：Buffer solution B；
Overall flow rate：100μL/min；
Purifying of poly (MPC-co-MBA) the organic polymer integral materials to inflammatory patients serum (a) and mouse blood plasma (b)
Result figure is as shown in figure 5, by Fig. 5 results it is found that the CRP in sample can be enriched with by the adsorbent, and other albumen cannot be inhaled
It is attached in the integral post, it was demonstrated that phospholipid Organic Polymer Monolithic Columns of the invention prepared can be successfully applied in actual sample
The purifying of CRP.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (10)
1. a kind of preparation method of phospholipid organic polymer integral material, it is characterised in that including following preparation process：
Phospholipid monomeric compound, crosslinking agent, pore-foaming agent and initiator are mixed, then pour into container after ultrasonic dissolution, degassing
Interior, the polymerisation at 40~70 DEG C of temperature or ultraviolet irradiation condition obtains the phospholipid organic polymer integral material；
The phospholipid monomeric compound refers to 2- (methacryloxy) ethyl-[N- (2- first such as 1 structure of following formula
Base acryloxy) ethyl] Phosphorylcholine compound, the single-stranded phosphatidyl choline compounds of 2 structure of formula, the double-strand of 3 structure of formula
Phosphatidyl choline compounds, the single-stranded phosphatidyl ethanol amine compounds of 4 structure of formula, the dichain phospholipids acyl of 5 structure of formula are ethanolaminated
Close object, the single-stranded phosphotidate of 6 structure of formula, the dichain phospholipids acid compound of 7 structure of formula, the single-stranded phosphatidyl of 8 structure of formula
Serine compound, at least one of dichain phospholipids acyl serine compound of 9 structure of formula, A is N or O elements in formula, n's
Range is 2~18；
2. a kind of preparation method of phospholipid organic polymer integral material according to claim 1, it is characterised in that：Institute
The crosslinking agent stated is N, in N'- methylene-bisacrylamides, ethylene glycol dimethacrylate, polyethyleneglycol diacrylate
Any one, the mass ratio of the phospholipid monomeric compound and crosslinking agent is (1:4)~(4:1).
3. a kind of preparation method of phospholipid organic polymer integral material according to claim 1, it is characterised in that：Institute
The pore-foaming agent stated is in isopropanol, tetrahydrofuran, dimethyl sulfoxide (DMSO), methanol, cyclohexanol, Isosorbide-5-Nitrae butanediol, n-dodecanol, water
The mass ratio of the arbitrary two kinds double base mixed systems formed, two of which pore-foaming agent is (1:5)~(5:1)；The phospholipid
The mass ratio of monomeric compound and pore-foaming agent is (1:6)~(1:1).
4. a kind of preparation method of phospholipid organic polymer integral material according to claim 1, it is characterised in that：Institute
The initiator stated is thermal initiator azodiisobutyronitrile or photoinitiator benzoin dimethylether or 2,2 '-azodiisobutyronitrile amidines
Any one in dihydrochloride, benzoyl peroxide, the double lauroyl of peroxidating, the addition of the initiator is phospholipid list
The 1% of body compound quality.
5. a kind of preparation method of phospholipid organic polymer integral material according to claim 1, it is characterised in that：Institute
The container stated is stainless steel tube, glass tube, capillary, solid phase extraction column, liquid transfer gun head, Solid Phase Extraction suction nozzle, magnetic Nano
Particle, lamellae, filter paper, filter membrane or glass monolith bottle.
6. a kind of preparation method of phospholipid organic polymer integral material according to claim 1, it is characterised in that：Institute
The time for stating polymerisation is 30~1440min；After the completion of the polymerisation, with methanol further rinse remove pore-foaming agent,
Unreacted monomer, crosslinking agent and oligomer.
7. a kind of phospholipid organic polymer integral material, it is characterised in that：Pass through claim 1~6 any one of them side
Method is prepared.
8. application of a kind of phospholipid organic polymer integral material in C reactive protein purifying described in claim 7.
9. a kind of phospholipid organic polymer integral material according to claim 8 answering in C reactive protein purifying
With, it is characterised in that the applying step is as follows：Phospholipid integral material is first used to buffer solution A balance a period of times, then is added
Enter the solution to be purified containing CRP, impurity is then eluted by buffer solution A, buffer solution B elutions is finally used to be inhaled by specificity
Attached CRP obtains C reactive protein after purification；The group of the buffer solution A become 10mM trishydroxymethylaminomethanes, 50~
400mM sodium chloride, 2~10mM calcium chloride, pH=8.0~8.5；The group of the buffer solution B becomes 10mM trihydroxy methyl amino
Methane, 50~400mM sodium chloride, 2~10mM disodium ethylene diamine tetraacetates, pH=8.0~8.5.
10. a kind of phospholipid organic polymer integral material according to claim 9 answering in C reactive protein purifying
With, it is characterised in that：The buffer solution A equilibration times are 0~60min；The solution to be purified containing CRP is containing CRP's
The serum or animal blood serum of standard protein solution, inflammatory patients.
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