CN115869925A - Polymer microsphere, preparation based on reversed phase suspension polymerization method and application thereof - Google Patents
Polymer microsphere, preparation based on reversed phase suspension polymerization method and application thereof Download PDFInfo
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Abstract
The invention relates to a polymer microsphere, and preparation and application thereof based on an inverse suspension polymerization method. Specifically, vinylphosphonic acid is used as a functional monomer, N, N-methylene bisacrylamide is used as a cross-linking agent, sodium persulfate is used as an initiator, water and N-heptane are used as dispersion media, and an inverse suspension polymerization method is adopted to prepare the phosphate radical functionalized polymer microspheres through free radical polymerization reaction; the microspheres do not need to be derived and can be directly mixed with titanium ions (Ti) 4+ ) Chelation was carried out to prepare Ti in only two steps 4+ -polymeric microspheres. The microsphere can be used as an adsorbent of immobilized metal ion affinity chromatography for separation and enrichment of phosphopeptides in biological samples. Ti prepared by the method 4+ The polymer microspheres have simple production process. The phosphate radical functionalized polymer microspheres can be prepared by only one step, and a complex grafting or derivation process is not needed before chelation with titanium ions, so that the preparation process is greatly simplified, and the preparation method is suitable for large-scale preparation.
Description
Technical Field
The invention relates to Ti prepared based on an inverse suspension polymerization method 4+ The polymer microsphere and the application thereof in the aspect of phosphopeptide separation and enrichment, in particular to a phosphate functionalized polymer microsphere obtained by performing reversed phase suspension polymerization on a functional monomer of vinylphosphonic acid and a cross-linking agent of N, N-methylene bisacrylamide in a dispersion medium consisting of water and N-heptane under the initiation action of an initiator of sodium persulfate. The microspheres can continue to chelate Ti due to the existence of the phosphate groups 4+ In two steps to obtain Ti 4 + -polymeric microspheres. Ti 4+ The polymer microsphere can be directly used as an adsorbent for immobilized metal ion affinity chromatography and is used for separating and enriching phosphopeptide in biological samples.
Background
Protein phosphorylation is considered to be one of the posttranslational modifications of proteins, which are involved in most of the life processes, such as cell division and differentiation, growth and apoptosis, and intercellular signal recognition and transmission, and also can be used as a marker for abnormal expression of cancer and other diseases (Nanomaterials in proteomics. Adv Funct material.n.sun et al.2019, 29. Abnormal protein phosphorylation often leads to the development of several diseases and even cancers (Magnetic metal phenyl networks: expanding the application of a promoting nanoprobe to phosphoproteomics research. Che. Commun. H. Chu et al.2020, 56. Therefore, the analysis and detection of the state and change of phosphorylated proteins/polypeptides are helpful for further understanding the mechanism of action thereof in the body, and are of great importance in the field of life science. Currently, the "shotgun" analysis strategy is one of the main methods for phosphoproteomics research, and mass spectrometry is the most commonly used analytical tool in phosphoproteins/polypeptides (MALDI TOF MS-based application of tandem enrichment strategy in phosphoproteomics. Analytical test report. Zhang Chuan quiet et al, 2017,36, 1064-1068.. However, direct MS analysis is difficult due to low phosphopeptide content, low ionization efficiency and interference of non-phosphorylated peptides in protein enzymatic hydrolysate (Robust phosphopeptide enzyme using microbial-based immobilized titanium (IV) affinity chromatography. Nat protocol. H. Zhou et al 2013,8 461-480.. Therefore, there is a need for separation and enrichment prior to analysis to improve the accuracy and sensitivity of the analysis.
To date, various methods for enrichment of phosphorylated peptides have been developed, including immunoaffinity capture, metal oxide affinity chromatography, immobilized metal ion affinity chromatography, and ion exchange chromatography, among others. Among them, ti found by Zou Hanfa research team 4+ And Zr 4+ With specific interaction between phosphorylated protein/polypeptide, phosphate group as Zr 4+ The new generation of immobilized metal ion affinity chromatography stationary phase of chelating ligand can obviously improve the enrichment capacity of phosphorylated protein/polypeptide (phosphorylated peptide fragment separation and enrichment method research progress. Analytical test report. Li Sha, et al.2020, 39. Because the immobilized metal ion affinity chromatography has unique selectivity and enrichment specificity to the phosphorylated peptide, the method is one of the most popular phosphorylated peptide enrichment methods at present.
Currently, the immobilized metal ion affinity chromatography materials mainly include nanoparticles, polymer microspheres, bulk monolithic materials, membranes and the like, but these materials are prepared by derivatizing or grafting functional groups onto a substrate, for example, the immobilized metal ion affinity chromatography adsorbent prepared by Zou Hanfa research team requires that polystyrene microspheres are prepared first, then epoxy, amino and phosphate functional groups are grafted, and finally titanium ions are chelated, and the immobilized metal ion affinity chromatography material can be obtained through five steps of reactions, which have complex preparation procedures, multiple steps and long time consumption, and are not suitable for large-scale preparation, so that the novel preparation method with simple exploration process is always a hotspot of research (focus of biological adsorption for immobilized N-polysaccharides and polypeptides, animal Chim acta. R. Tang et al.2021,1144: 111-120.).
Disclosure of Invention
The invention aims to provide Ti prepared based on an inverse suspension polymerization method 4+ Polymer microspheres, in particular phosphate-functionalized polymer microspheres prepared by inverse suspension polymerization and further mixed with Ti 4+ Chelating to obtain Ti 4+ -polymeric microspheres. The microspheres can be directly used as immobilized metal ion affinity chromatography adsorbents for separating and enriching phosphopeptides in biological samples.
To achieve the above object, the following process can be performed:
1) Preparation of phosphate functionalized polymer microspheres: adding 200-1200 mg of vinyl phosphonic acid into a container, adding N, N-methylene bisacrylamide into 1-20 mL of water according to the molar ratio of the N, N-methylene bisacrylamide to the vinyl phosphonic acid of 1.5/1-0.4/1 to form a uniform and transparent solution, and adjusting the pH of the solution to 6-8 by using 1M sodium hydroxide solution; continuing to add a stabilizer and n-heptane, wherein the volume ratio of the n-heptane to the water phase is 2/1-8/1, the stabilizer comprises span 80 and tween 80, 300-1500 mg of span 80 is added, the mass ratio of the tween 80 to the span 80 is 1/1-2/3, and the mixture is ultrasonically mixed uniformly; then adding 50-300 mg of initiator sodium persulfate, and mechanically stirring for 10-40 min at the rotating speed of 100-300 r/min; then raising the temperature to 60-80 ℃, and continuing stirring for 4-8 hours to complete the polymerization reaction; finally, washing the obtained microsphere material with an ethanol/water (1/1-1/2,v/v) mixed solvent for 3-5 times, and carrying out vacuum drying at the temperature of 60-80 ℃ for 8-24 hours to obtain phosphate radical functionalized polymer microspheres;
2)Ti 4+ preparation of polymeric microspheres: dispersing 1-2 g of phosphate radical functionalized polymer microspheres and titanium sulfate in 10-50 mL of water according to the mass ratio of 1/5-1/50, incubating for 6-12 hours at room temperature, washing the obtained material with an ethanol/water (1/1-1/2,v/v) mixed solvent for 3-5 times, and vacuum drying for 8-24 hours at the temperature of 60-80 ℃ to obtain Ti 4+ -polymeric microspheres.
The invention has the following advantages:
(1) The phosphate radical functionalized polymer microspheres can be obtained by one-step reaction based on the reversed-phase suspension polymerization method, the production process is simple, the reaction conditions are mild, and the method is suitable for large-scale preparation;
(2) The phosphate radical functionalized polymer microsphere does not need late grafting or derivation and can be directly mixed with Ti 4+ Chelating to obtain Ti 4+ -polymeric microspheres;
(3)Ti 4+ the polymer microspheres are used as an adsorbent for immobilized metal ion affinity chromatography and have high-efficiency separation and enrichment capacity on phosphopeptides in biological samples.
Drawings
FIG. 1Ti 4+ Schematic preparation of polymeric microspheres.
FIG. 2Ti 4+ -helium ion scanning electron micrographs of polymeric microspheres. a: example 1,b: example 2,c: comparative example 2,d comparative example 3.
FIG. 3 is a comparison graph of Fourier transform-infrared spectra of phosphate functionalized polymer microspheres and monomers prepared therefrom.
FIG. 4Ti 4+ The full spectrum of X-ray photoelectron spectrum of the polymer microsphere and the high resolution spectrum of Ti 2 p.
FIG. 5Ti 4+ Nitrogen adsorption/desorption curves of polymeric microspheres and their pore size distribution profiles. a. c: nitrogen adsorption/desorption curve, b, d: the aperture profile.
FIG. 6Ti 4+ Mass spectrum comparison chart of polymer microsphere to beta-casein and bovine serum albumin (1/100, mass ratio) before and after enrichment. a: before enrichment, b: ti 4+ -after enrichment of the polymeric microspheres I; c: ti 4+ After enrichment of the polymer microspheres II. * Represents phosphopeptide, # represents dephosphorylated fragment
FIG. 7 is a mass spectrum of enriched beta-casein and bovine serum albumin (1/100, mass ratio). a: commercial Ti 4+ -enrichment of affinity chromatography microspheres, b: ti 4+ After enrichment of the polymeric microspheres III, c: ti 4+ -polymer microspheres IV enriched with phosphopeptides, # represents dephosphorylated debris.
Detailed Description
Example 1Ti 4+ Preparation and use of Polymer microspheres I
Ti 4+ Preparation of polymeric microspheres I:
1) And (3) preparing phosphate radical functionalized polymer microspheres I. Adding 1080mg of vinyl phosphonic acid into a container, adding N, N-methylene bisacrylamide into 12mL of water according to the molar ratio of the N, N-methylene bisacrylamide to the vinyl phosphonic acid being 1/1 to form a uniform and transparent solution, and adjusting the pH value of the solution to be 7 by using 1M sodium hydroxide solution; continuing to add a stabilizer and n-heptane, wherein the volume ratio of the n-heptane to the water phase is 5/1, the stabilizer comprises span 80 and tween 80, adding 1200mg of span 80, and the mass ratio of tween 80 to span 80 is 2/3, and ultrasonically mixing uniformly; then 200mg of initiator sodium persulfate is added, and mechanical stirring is carried out for 30min at the rotating speed of 300 r/min; then raising the temperature to 70 ℃, and continuing stirring for 6 hours to complete the polymerization reaction; finally, washing the obtained microsphere material with an ethanol/water (1/1, volume ratio) mixed solvent for 3 times, and carrying out vacuum drying at the temperature of 60 ℃ for 12 hours to obtain a phosphate radical functionalized polymer microsphere I;
2)Ti 4+ preparation of polymeric microspheres I. Dispersing 1g of polymer microsphere I containing phosphate radical functional groups and titanium sulfate in 40mL of water according to the mass ratio of 1/10 of the polymer microsphere to the titanium sulfate, incubating for 8 hours at room temperature, washing the obtained material with an ethanol/water (1/1, volume ratio) mixed solvent for 5 times, and drying for 12 hours at 60 ℃ in vacuum to obtain Ti 4+ -polymeric microspheres I.
Ti 4+ Use of polymeric microspheres I:
preparation of beta-casein (beta-casein) enzymatic hydrolysate sample: 2.0mg of beta-casein was dissolved in 200. Mu.L of a solution containing 6M guanidine hydrochloride and 100mM NH 4 HCO 3 In an aqueous solution of a denaturing agent. 1.4mL of Tris-HCl buffer was added. Adding trypsin according to the enzyme-protein mass ratio of 1.
Preparation of Bovine Serum Albumin (BSA) enzymatic hydrolysate sample: 2.0mg of BSA were dissolved in 1.0mL of a solution containing 8M urea and100mM NH 4 HCO 3 in an aqueous solution of a denaturing agent. Then 20 μ L of 1M aqueous solution of dithiothreitol is added, and after centrifugation at 10000r/min for 20min, the temperature is kept constant at 37 ℃ for 2h. 7.4mg of iodoacetamide was further added in the dark, and the reaction was carried out for 35min in the dark. After removal, 7.0mL of Tris-HCl buffer was added. Adding trypsin according to the enzyme-protein mass ratio of 1.
Enrichment process of phosphorylated peptide: first, 200. Mu.L of a sample solution (ACN/H) was added 2 O/TFA,80/14/6,v/v/v) balance Ti obtained as described above 4+ Polymer microspheres I were shaken twice at room temperature for 15min each time, centrifuged and the supernatant removed. mu.L of a sample solution (ACN/H) containing a 1/100 mass ratio of beta-casein/BSA (m/m) enzymatic hydrolysate 2 O/TFA,80/14/6,v/v/v) with 100. Mu.L 0.1% aqueous TFA Ti was added as 1/1 (v/v) solution 4+ Shaking and loading the polymer microsphere material for 30min at room temperature, centrifuging and removing supernatant. Then, the eluent A (ACN/H) containing 400mM NaCl was used 2 O/TFA,50/44/6,v/v/v) elution twice (200. Mu.L. Times.2 times), each time with shaking for 15min, centrifugation, and removal of supernatant to remove non-phosphopeptides and other impurities. Adding leacheate B (ACN/H) 2 O/TFA,30/69.9/0.1, v/v/v) was rinsed twice (200. Mu.L. Times.2 times) to remove the salt. Followed by addition of 100. Mu.L of 10% by mass NH 3 .H 2 And shaking the eluent at room temperature for 15min, centrifuging, and taking the supernatant to analyze by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS).
Product characterization
As shown in FIG. 2a, the material is spherical with a particle size of 10 to 22 μm by a helium ion scanning electron microscope.
The infrared spectrum of the phosphate functionalized polymer microsphere prepared in example 1 and the precursor thereof is shown in FIG. 3, and the wave number in the monomer vinylphosphonic acid spectrum is 1108cm -1 ，1023cm -1 Respectively correspond to-PO 3 Middle P = stretching vibration peak of O bond and P — O bond stretching vibration peak. These peaks are also present in the ir spectrum of the phosphate functionalized polymeric microspheres. Crosslinking agent N, N-methylene-bisThe wave number in an acrylamide spectrogram is 1655cm -1 And 1538cm -1 The absorption peaks of (a) correspond to an amide bond I-C = O stretching vibration peak and an amide bond II-N-H stretching vibration peak respectively, and the peaks are also present in an infrared spectrogram of the phosphate radical functionalized polymer microsphere. However, stretching vibration of C = C bond in two precursor spectra (1625 cm) -1 ) No observation was made in the spectra of the phosphate functionalized polymeric microspheres, indicating that C = C had been consumed during the polymerization reaction. These analysis results show that the phosphate radical functionalized polymer microspheres are prepared from precursors of vinylphosphonic acid and N, N-methylene bisacrylamide through a free radical reaction of ethylene.
FIG. 4 is Ti 4+ Experimental results of the full spectrum of the X-ray photoelectron spectrum of the polymer microsphere and the high resolution spectrum of Ti 2 p. Ti can be clearly observed in the whole spectrum 4+ The polymeric microspheres contained an absorption peak of C, O, P and Ti atoms (fig. 4 a). In the Ti 2p high resolution spectrum, the absorption peaks of 458.7eV and 464.4eV correspond to the 3/2p and 1/2p orbits of Ti, respectively (FIG. 4 b). Thus, it is known that Ti 4+ The polymer microspheres are successfully reacted with Ti through phosphate radicals on the surfaces of the microspheres 4+ Prepared by chelation.
FIG. 5 shows the characterization results of nitrogen physical adsorption/desorption experiment, ti 4+ The specific surface area of the polymeric microspheres I is 204.9m 2 g -1 Mesopores with an average pore diameter of 15.5nm (FIG. 5 b).
Product application
Ti 4+ The polymer microspheres I can be used as an adsorbent for immobilized metal ion affinity chromatography to separate and enrich phosphopeptides in a sample. FIG. 6 is a comparison graph of mass spectra before and after enrichment of the mixed enzymatic hydrolysate of beta-casein and bovine serum albumin. As shown in FIG. 6a, the peak signals with higher intensity in the spectra before enrichment are all non-phosphopeptide signal peaks, phosphopeptide signals are almost all inhibited, and no significant phosphopeptide signals are observed. And use Ti 4+ After enrichment of the polymer microsphere I, as shown in FIG. 6b, the non-phosphopeptide peaks were significantly reduced and the phosphopeptide peak signal was significantly enhanced, and 3 typical phosphopeptide signal peaks (2061, 2556,3121, m/z) and dephosphorylated fragment peaks (1963, 2458,3023, m/z) were detected) Description of the Ti 4+ The polymer microspheres I have high enrichment effect and selectivity on phosphopeptides.
Example 2Ti 4+ Preparation and application of polymer microspheres II
Ti 4+ Preparation of polymeric microspheres II:
in the preparation process of the phosphate radical functionalized polymer microsphere II, the mass of the functional monomer vinyl phosphonic acid is reduced to 864mg, and the rest of the preparation conditions and the titanium ion chelating process are the same as those in example 1Ti 4+ Preparation of polymeric microspheres I.
Obtained Ti 4+ Use of Polymer microspheres II also as in example 1Ti 4+ -use of polymeric microspheres I.
Product characterization
Ti 4+ Helium ion scanning electron microscope of the polymer microspheres II As shown in FIG. 2b, the material is spherical and has a particle size of 15-35 μm. The results of the nitrogen physical adsorption/desorption experiments (FIG. 5 a) show that Ti 4+ Polymer microspheres II specific surface area ratio Ti 4+ Polymer microspheres I Low, value 158.0m 2 g -1 . The pore size distribution shows that the microspheres and Ti 4+ Polymer microspheres II, like these, also contain mesopores with an average pore diameter of 14.2nm (FIG. 5 b). This is probably because the decrease of the content of the functional monomer vinylphosphonic acid in the preparation process of the polymer microsphere-II leads to the increase of the relative content of the cross-linking agent N, N-methylene-bisacrylamide in the reaction solution, the increase of the cross-linking degree and the decrease of the comparative area.
Product application
As shown in FIG. 6c, ti is used 4+ When the polymer microspheres II are adsorbents, 3 pieces of phosphopeptides (2061, 2556,3121, m/z) with characteristics can be enriched from the mixed enzymolysis liquid of beta-casein and bovine serum albumin (1/100, mass ratio), which shows that the polymer microspheres II have better enrichment efficiency and selectivity. However, ti prepared in accordance with example 1 4+ The enrichment effect of the polymer microspheres I is slightly higher than that of the non-phosphopeptides, which indicates that Ti 4+ The polymer microspheres II are less selective for phosphopeptides than Ti 4+ -polymeric microspheres I. This may be due to the functional monomers used in their preparationLess vinylphosphonic acid results in a decrease in phosphate binding sites available after spheronization and hence a lower number of titanium ions available for chelation.
Comparative example 1
Enrichment of phosphopeptides using commercial immobilized metal ion affinity chromatography microspheres
For comparison with examples, commercial Ti was purchased from Bailingwei Tech Co Ltd 4+ Affinity microsphere material (SPE-Ti-IMAC, particle size 40-100 μm), under the same conditions (same as example 1 Ti) 4+ Application of polymer microspheres I) to the enrichment of phosphopeptides in the mixed enzymatic hydrolysate of beta-casein and bovine serum albumin. The results are shown in FIG. 7. Although 3 characteristic phosphopeptide signals were detectable (2061, 2556,3121, m/z) and interference from non-phosphopeptides was almost negligible, their signal intensity was significantly lower than in examples 1 and 2. This indicates that although commercial SPE-Ti-IMAC microspheres have good phosphopeptide selectivity, the enrichment efficiency is slightly lower.
Comparative example 2
Ti 4+ Preparation of polymeric microspheres III:
in the preparation process of the phosphate radical functionalized polymer microsphere III, 1M sodium hydroxide solution is replaced by ammonia water with the same molar mass concentration, and the rest preparation conditions and the titanium ion chelation process are the same as those of example 1, ti 4+ Preparation of polymeric microspheres I.
Obtained Ti 4+ Use of polymeric microspheres III also in example 1Ti 4+ -use of polymeric microspheres I.
Product characterization
Ti 4+ Helium ion scanning Electron microscopy of Polymer microspheres III As shown in FIG. 2c, the material is amorphous and non-spherical. The results of the nitrogen physical adsorption/desorption experiments (FIG. 5 c) show that Ti 4+ Polymer microspheres III specific surface area ratio Ti 4+ The polymer microspheres I and II are both low and have a value of 11.6m 2 g -1 . The pore size distribution indicates that the material is mesoporous with an average pore size of 5.9nm (fig. 5 d). This is probably because it does not self-sphere, is amorphous and gives insufficient Ti yield 4+ -polymersHalf of microspheres I and II, which do not form a cross-linked structure, result in almost no specific area.
Product application
As shown in FIG. 7b, ti was used 4+ When the polymer microspheres III are adsorbents, 3 pieces of phosphopeptides (2061, 2556,3121, m/z) with characteristics can be enriched from the mixed enzymatic hydrolysate of beta-casein and bovine serum albumin (1/100, mass ratio), which shows that the polymer microspheres III have enrichment effect on phosphopeptides. However, ti prepared in accordance with examples 1 and 2 4+ Compared with the enrichment effect of the polymer microspheres I, II, the interference of non-phosphopeptides is more, and the signal intensity is lower, which indicates that Ti 4+ The selectivity and enrichment efficiency of the polymer microspheres III on phosphopeptides are inferior to those of Ti 4+ -polymeric microspheres I. This is probably due to the fact that the morphology is amorphous, there is little specific surface area, few phosphate binding sites are available and therefore the amount of titanium ions available for sequestration is low.
Comparative example 3
Ti 4+ Preparation of polymeric microspheres IV:
in the preparation process of the phosphate radical functionalized polymer microsphere IV, a 1M sodium hydroxide solution is directly replaced by a titanium sulfate solution with the same molar mass concentration without a step of chelating titanium ions, and the rest preparation conditions are the same as those of example 1, ti 4+ Preparation of polymeric microspheres I.
Obtained Ti 4+ Use of Polymer microspheres IV also as in example 1Ti 4+ -use of polymeric microspheres I.
Product characterization
Ti 4+ Helium ion scanning Electron microscopy of Polymer microspheres IV As shown in FIG. 2d, the material is amorphous and non-spherical. The results of the nitrogen physical adsorption/desorption experiments (FIG. 5 c) show that Ti 4+ Polymer microspheres IV specific surface area ratio Ti 4+ The polymer microspheres I, II are all low, with a value of 12.1m 2 g -1 Same as Ti 4+ The polymeric microspheres III are comparable and have essentially no specific surface area. The pore size distribution indicated that it contained mesopores with an average pore size of 6.8nm (FIG. 5 d). This is probably because the titanium sulfate was added directly to the solution, no microspheres were formed, and the product was obtainedThe ratio is equal to Ti 4+ The polymer microspheres III are comparable and do not form a cross-linked structure, resulting in almost no specific area.
Product application
As shown in FIG. 7c, use is made of Ti 4+ When the polymer microspheres IV are used as an adsorbent, only 2 phosphopeptides (2061, 3121, m/z) with characteristics can be enriched from the mixed enzymatic hydrolysate of beta-casein and bovine serum albumin (1/100, mass ratio), which indicates that the efficiency of enriching phosphopeptides is low. And Ti prepared in example 1 4+ Compared with the enrichment effect of the polymer microspheres I, the interference of non-phosphopeptide is very much, the signal intensity of the phosphopeptide is suppressed by the signal intensity of the non-phosphopeptide, so that the signal intensity of the phosphopeptide is less than 1000, which indicates that Ti 4+ The selectivity and enrichment efficiency of the polymer microspheres IV on phosphopeptides are inferior to those of Ti 4+ -polymeric microspheres I. This is probably due to the fact that the morphology is amorphous, there is little specific surface area, few phosphate binding sites are available and therefore the amount of titanium ions available for sequestration is low.
The above results show that Ti prepared by the present invention 4+ The polymer microspheres I show high enrichment efficiency and selectivity for phosphopeptides. The preparation method is simple to operate. The phosphate radical functionalized polymer microspheres can be synthesized by reverse phase suspension syncopation and synthesis, and can be directly combined with Ti without secondary modification 4 Chelating to prepare Ti for enriching phosphopeptide 4 + The polymer microspheres greatly simplify the preparation process and are suitable for large-scale preparation.
Claims (6)
1. A polymer microsphere and preparation based on an inverse suspension polymerization method are disclosed, vinyl Phosphonic Acid (VPA) is used as a functional monomer, N, N-Methylene Bisacrylamide (MBA) is used as a cross-linking agent, a water-in-oil system is formed in the presence of a stabilizer, inverse suspension polymerization is adopted, and phosphate radical functionalized polymer microspheres are prepared through radical polymerization reaction; the microspheres are further mixed with titanium ions (Ti) 4+ ) Chelating to obtain Ti for phosphopeptide enrichment 4+ -polymeric microspheres.
2. The method for producing polymer microspheres according to claim 1, wherein:
sodium persulfate is used as a free radical initiator;
taking non-ionic surfactant span 80 and tween 80 as a mixed stabilizer, wherein the hydrophilic-lipophilic balance value of the mixed stabilizer is 3.0-9.0;
the mixed solvent of n-heptane and water is used as continuous medium.
3. The production method according to claim 1 or 2, characterized in that: ti 4+ -the polymeric microspheres are prepared as follows:
1) Preparation of phosphate functionalized polymer microspheres: adding 200-1200 mg of vinyl phosphonic acid into a container, adding N, N-methylene bisacrylamide into 1-20 mL of water according to the molar ratio of the N, N-methylene bisacrylamide to the vinyl phosphonic acid of 1.5/1-0.4/1 to form a uniform and transparent solution, and adjusting the pH of the solution to 6-8 by using 1M sodium hydroxide solution; continuing to add a stabilizer and n-heptane, wherein the volume ratio of the n-heptane to the water phase is 2/1-8/1, the stabilizer comprises span 80 and tween 80, 300-1500 mg of span 80 is added, the mass ratio of the tween 80 to the span 80 is 1/1-2/3, and the mixture is ultrasonically mixed uniformly; then adding 50-300 mg of initiator sodium persulfate, and mechanically stirring for 10-40 min at the rotating speed of 100-300 r/min; then raising the temperature to 60-80 ℃, and continuing stirring for 4-8 hours to complete the polymerization reaction; finally, washing the obtained microsphere material with an ethanol/water (1/1-1/2,v/v) mixed solvent for 3-5 times, and carrying out vacuum drying at the temperature of 60-80 ℃ for 8-24 hours to obtain phosphate radical functionalized polymer microspheres;
2)Ti 4+ preparation of polymeric microspheres: dispersing 1-2 g of phosphate radical functionalized polymer microspheres and titanium sulfate in 10-50 mL of water according to the mass ratio of 1/5-1/50, incubating for 6-12 hours at room temperature, washing the obtained material with an ethanol/water (1/1-1/2,v/v) mixed solvent for 3-5 times, and vacuum drying for 8-24 hours at the temperature of 60-80 ℃ to obtain Ti 4+ -polymeric microspheres.
4. A polymeric microsphere obtainable by the process according to any one of claims 1 to 3.
5. Use of the polymeric microspheres of claim 4, wherein: the microsphere can be used as an adsorbent or a stationary phase of immobilized metal ion affinity chromatography, and is used for separating and enriching phosphopeptides in biological samples.
6. Use according to claim 5, characterized in that: the biological sample is one or more than two of human and/or animal body fluid, tissue enzymolysis liquid, cell enzymolysis liquid and the like.
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| CN116715805B (en) * | 2023-08-01 | 2023-10-27 | 苏州大学 | Multifunctional gel microsphere rich in bisphosphonic acid groups and preparation method and application thereof |
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