CN113058576B - Hollow core-shell structure nano magnetic microsphere, preparation method and application thereof - Google Patents
Hollow core-shell structure nano magnetic microsphere, preparation method and application thereof Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 104
- 239000011258 core-shell material Substances 0.000 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 134
- 239000000243 solution Substances 0.000 claims abstract description 33
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 28
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims abstract description 24
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims abstract description 22
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims abstract description 17
- 239000004472 Lysine Substances 0.000 claims abstract description 16
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims abstract description 14
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000007853 buffer solution Substances 0.000 claims abstract description 12
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 9
- BPSIOYPQMFLKFR-UHFFFAOYSA-N trimethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane Chemical compound CO[Si](OC)(OC)CCCOCC1CO1 BPSIOYPQMFLKFR-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000004593 Epoxy Substances 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 34
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- 102000004196 processed proteins & peptides Human genes 0.000 claims description 17
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 16
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- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 16
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
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- 229910021578 Iron(III) chloride Inorganic materials 0.000 abstract 1
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
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- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
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- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
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- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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- AISMNBXOJRHCIA-UHFFFAOYSA-N trimethylazanium;bromide Chemical compound Br.CN(C)C AISMNBXOJRHCIA-UHFFFAOYSA-N 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
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- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
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- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
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- B01J20/28004—Sorbent size or size distribution, e.g. particle size
- B01J20/28007—Sorbent size or size distribution, e.g. particle size with size in the range 1-100 nanometers, e.g. nanosized particles, nanofibers, nanotubes, nanowires or the like
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- B01J20/28016—Particle form
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Abstract
The present disclosure provides a hollow core-shell structure nano magnetic microsphere, a preparation method and an application thereof. The method comprises the following steps: providing Fe 3 O 4 A nano magnetic microsphere; it reacts with hexadecyl trimethyl ammonium bromide and tetraethyl silicate in ethanol to obtain Fe with a hollowed-out core-shell structure 3 O 4 @mSiO 2 The method comprises the steps of carrying out a first treatment on the surface of the Dispersing in ethanol by ultrasonic wave, adding glycidyl oxypropyl trimethoxy silane to obtain Fe 3 O 4 @mSiO 2 A @ GPS; dispersing in tetrahydrofuran by ultrasonic wave, adding NH 2 ‑PEG n ‑NH 2 Obtaining the amino modified Fe 3 O 4 @mSiO 2 @NH 2 A nano magnetic microsphere; ultrasonic dispersing in 2, 4-epoxy hexacyclic ring or tetrahydrofuran, adding N, N-carbonyl diimidazole to react, dispersing in alkaline buffer solution, adding N α ,N α -di (carboxymethyl) -L-lysine to give carboxylic acid modified Fe 3 O 4 @mSiO 2 @NTA; dispersing in ferric chloride solution by ultrasonic wave to obtain the final product.
Description
Technical Field
The disclosure relates to the technical field of organic materials and analysis, in particular to a hollow core-shell structure nano magnetic microsphere, a preparation method and application thereof.
Background
The nano magnetic material is a new material developed in recent years and is widely applied to the fields of nuclear magnetic imaging, catalysis, sensing, enrichment materials and the like.
However, there are three problems in the process of functionalizing magnetic nanoparticles, (1) loss of dispersibility, i.e., small nanoparticles tend to aggregate to form large particles to reduce surface energy; (2) Loss of magnetic properties, i.e. bare magnetic nanoparticles, in particular Fe 3 O 4 And gamma-Fe 2 O 3 Is easy to oxidize in air, and the acid environment in the process of enriching the phosphorylated peptide can damage the integrity of the magnetic nano microsphere; (3) The magnetic nano microsphere has longer surface functionalization time, harsh synthesis conditions and is not suitable for large-scale popularization.
Disclosure of Invention
In view of the above, the present disclosure aims to provide a hollow core-shell structure nano magnetic microsphere, and a preparation method and application thereof.
Based on the above objects, an embodiment of the present disclosure provides a method for preparing a hollow core-shell structure nano magnetic microsphere, including:
providing Fe 3 O 4 A nano magnetic microsphere;
fe is added to 3 O 4 Nanometer magnetic microsphere, hexadecyl trimethyl ammonium bromide and tetraethyl silicate react in ethanol in alkaline environment to obtain Fe with hollowed-out core-shell structure 3 O 4 @mSiO 2 ；
Fe is added to 3 O 4 @mSiO 2 Dispersing in ethanol by ultrasonic wave, adding glycidyl oxypropyl trimethoxy silane, reacting in alkaline environment, and obtaining Fe at normal temperature 3 O 4 @mSiO 2 @GPS；
Fe is added to 3 O 4 @mSiO 2 Dispersing the @ GPS ultrasonic wave in tetrahydrofuran, adding NH 2 -PEG n -NH 2 Reacting to obtain amino modified Fe 3 O 4 @mSiO 2 @NH 2 A nano magnetic microsphere;
fe is added to 3 O 4 @mSiO 2 @NH 2 Ultrasonic separation of nano magnetic microspheresDispersing in 2, 4-epoxy hexacyclic ring or tetrahydrofuran, adding N, N-carbonyl diimidazole for reaction; dispersing the obtained product in alkaline buffer solution, adding N α ,N α -di (carboxymethyl) -L-lysine to give carboxylic acid modified Fe 3 O 4 @mSiO 2 @NTA；
Modification of carboxylic acid with Fe 3 O 4 @mSiO 2 Ultrasonic dispersion of @ NTA in ferric trichloride solution to obtain Fe 3 O 4 @mSiO 2 @NTA@Fe 3+ A nano magnetic microsphere.
In some embodiments, the Fe 3 O 4 The nano magnetic microsphere is prepared from 0.2-0.3M ferric trichloride by a hot solvent reduction method; the mass ratio of the cetyl trimethyl ammonium bromide to the tetraethyl silicate is 1: 1-1:3.
In some embodiments, the reaction yields Fe in a hollowed-out core-shell structure 3 O 4 @mSiO 2 The method specifically comprises the following steps:
fe is added to 3 O 4 The nanometer magnetic microspheres are ultrasonically dispersed in ethanol;
sequentially adding cetyl trimethyl ammonium bromide, tetraethyl silicate and ammonia water, and reacting for 2-6 hours; respectively cleaning the products obtained by the reaction for 2-3 times by adopting water and ethanol in turn to obtain Fe with a core-shell structure 3 O 4 @SiO 2 ；
Fe is added to 3 O 4 @SiO 2 Ultrasonic dispersing in ethanol or acetone and refluxing for 10-20 hours to obtain Fe with hollow core-shell structure 3 O 4 @mSiO 2 。
In some embodiments, the resulting Fe 3 O 4 @mSiO 2 In the step of @ GPS, the volume of the ethanol is 50-80 ml; the alkaline environment is provided for ammonia water, the final concentration of the ammonia water is 0.5-1.5mM, the volume of the glycidyl oxypropyl trimethoxysilane is 15-30ml, and the final concentration is 0.50M.
In some embodiments, the resulting amine-modified Fe 3 O 4 @mSiO 2 @NH 2 In the step of nano magnetic microsphere, the NH 2 -PEG n -NH 2 The molar ratio of the catalyst to the tetraethyl silicate is 1:1 to 4:1, the polymerization degree n of the ethylene glycol is 2-10, the reaction time is 6-8 hours, and the reaction temperature is 50 ℃.
In some embodiments, the resulting carboxylic acid-modified Fe 3 O 4 @mSiO 2 In the step @ NTA, the molar ratio of N, N-carbonyldiimidazole to tetraethyl silicate is 1:1 to 4:1, fe 3 O 4 @mSiO 2 @NH 2 The reaction time of the nano magnetic microsphere and N, N-carbonyl diimidazole is 1-2 hours.
In some embodiments, the resulting carboxylic acid-modified Fe 3 O 4 @mSiO 2 In the step @ NTA, the pH of the alkaline buffer solution is 9-11, and the pH of the alkaline buffer solution is N α ,N α -molar ratio of di (carboxymethyl) -L-lysine to the tetraethyl silicate 1.2:1-2:1, the Fe 3 O 4 @mSiO 2 @NH 2 Nanometer magnetic microsphere and N α ,N α The reaction time of the di (carboxymethyl) -L-lysine is 4-10 hours, and the reaction temperature is 4 ℃.
In some embodiments, the resulting Fe 3 O 4 @mSiO 2 @NTA@Fe 3+ In the step of the nano magnetic microsphere, the ferric trichloride solution is acetic acid solution of ferric trichloride, the molar quantity of the ferric trichloride is 0.8-1.2M, and the reaction time is 5-8 hours.
The embodiment of the disclosure also provides a hollow core-shell structure nano magnetic microsphere, which is prepared by the preparation method according to any one of the previous technical schemes.
The disclosure provides a preparation method of the hollow core-shell structure nano magnetic microsphere according to any one of the technical schemes or application of the hollow core-shell structure nano magnetic microsphere prepared by the preparation method in specific enrichment or purification of trace phosphorylated polypeptides.
From the above, it can be seen that the method for preparing the hollow core-shell structure nano magnetic microsphere provided by the present disclosure provides Fe 3 O 4 A nano magnetic microsphere; fe is added to 3 O 4 Nanometer magnetic microsphere and hexadecaneThe trimethyl ammonium bromide and the tetraethyl silicate react in ethanol in an alkaline environment to obtain Fe with a hollowed-out core-shell structure 3 O 4 @mSiO 2 The method comprises the steps of carrying out a first treatment on the surface of the Fe is added to 3 O 4 @mSiO 2 Dispersing in ethanol by ultrasonic wave, adding glycidyl oxypropyl trimethoxy silane, reacting in alkaline environment, and obtaining Fe at normal temperature 3 O 4 @mSiO 2 A @ GPS; fe is added to 3 O 4 @mSiO 2 Dispersing the @ GPS ultrasonic wave in tetrahydrofuran, adding NH 2 -PEG n -NH 2 Reacting to obtain amino modified Fe 3 O 4 @mSiO 2 @NH 2 A nano magnetic microsphere; fe is added to 3 O 4 @mSiO 2 @NH 2 The nano magnetic microsphere is dispersed in 2, 4-epoxy hexacyclic ring or tetrahydrofuran by ultrasonic, and N, N-carbonyl diimidazole is added for reaction; dispersing the obtained product in alkaline buffer solution, adding N α ,N α -di (carboxymethyl) -L-lysine to give carboxylic acid modified Fe 3 O 4 @mSiO 2 @NTA; modification of carboxylic acid with Fe 3 O 4 @mSiO 2 Ultrasonic dispersion of @ NTA in ferric trichloride solution to obtain Fe 3 O 4 @mSiO 2 @NTA@Fe 3+ A nano magnetic microsphere. The specific surface area of the finally obtained hollow core-shell structure nano magnetic microsphere particles can be remarkably improved, and the loading capacity of the surface functional groups is increased; can also improve Fe 3 O 4 Dispersibility of core particles while reducing Fe in acidic environment 3 O 4 Corrosion of the magnetic core; meanwhile, the flexibility of the nano material can be increased, and the negative effect that the phosphorylated polypeptide is not easy to elute due to the 'cavity effect' is reduced. The method has mild reaction conditions, greatly shortens the reaction time to 2 hours, and improves the preparation efficiency of the hollow core-shell structure nano magnetic microsphere. The obtained hollow core-shell structure nano magnetic microsphere can be used as an affinity reagent for carrying out specific enrichment and purification on trace phosphorylated polypeptides in complex biological samples, and has excellent specific enrichment and purification effects.
Drawings
In order to more clearly illustrate the technical solutions of the present disclosure or related art, the drawings required for the embodiments or related art description will be briefly described below, and it is apparent that the drawings in the following description are only embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort to those of ordinary skill in the art.
Fig. 1 is a schematic flow chart of a method for preparing a hollow core-shell structure nano magnetic microsphere according to an embodiment of the disclosure;
fig. 2 is a structural diagram of a hollow core-shell structure nano magnetic microsphere in a preparation process according to an embodiment of the disclosure;
FIG. 3 is an electron microscope image of the hollow-core-shell structure nano magnetic microsphere in example 1 during the preparation process; (a) Is Fe 3 O 4 Electron microscope image of nanometer magnetic microsphere; (b) Is Fe with hollowed surface 3 O 4 @mSiO 2 Electron microscope image of nanometer magnetic microsphere; (c) Is Fe modified by amino 3 O 4 @mSiO 2 @NH 2 Electron microscope image of nanometer magnetic microsphere; (d) Is Fe 3 O 4 @mSiO 2 @NTA@Fe 3+ Electron microscopy of the nano magnetic microsphere.
Fig. 4 is a graph of near infrared characterization results during the preparation process of the hollow-core-shell structure nano magnetic microsphere in example 1.
FIG. 5 is a sample of the LC-MS/MS detection of Fe used in example 2 3 O 4 @mSiO 2 @NTA@Fe 3+ The effect of enrichment of phosphorylated peptides in β -casein is schematically shown.
FIG. 6 (a) shows the core-shell nano-magnetic microsphere Fe in comparative example 1 3 O 4 @SiO 2 Electron microscopy after ether treatment;
fig. 7 is a comparison of fig. 6 (a) and fig. 3 (b).
Detailed Description
For the purposes of promoting an understanding of the principles and advantages of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same.
It should be noted that unless otherwise defined, technical or scientific terms used in the embodiments of the present disclosure should be given the ordinary meaning as understood by one of ordinary skill in the art to which the present disclosure pertains. The terms "first," "second," and the like, as used in embodiments of the present disclosure, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof, but does not exclude other elements or items. The terms "connected" or "connected," and the like, are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect.
Protein phosphorylation plays an important role in cell life activities, and is involved in gene transcription regulation, cell proliferation, development, differentiation, apoptosis, nerve signal transduction and other aspects. In cells, about 30% of the protein can undergo phosphorylation modification in vivo, with a ratio of serine phosphorylation, threonine phosphorylation, and tyrosine phosphorylation of 1800:200:1. the phosphorylation modification sites and change rules of the protein under different physiological conditions and in the disease occurrence process are researched, so that the protein is helpful to understand the participation of the protein in-vivo regulation and control mechanism and disease diagnosis and treatment.
With the increasing sensitivity and resolution of mass spectrometry, liquid chromatography-mass spectrometry has been dominant in high-throughput phosphorylated protein analysis. However, the phosphorylated proteins need to be enriched before mass spectrometry, so that the relative abundance of the phosphorylated proteins is improved, and the influence of non-phosphorylated proteins is reduced. At present, the enrichment of the phosphorylated proteins has the problems that the polypeptide with unknown phosphorylated sites cannot be enriched, the selectivity to the phosphorylated peptide segments is not good enough, and the like.
The applicant provides a hollow-core-shell structure magnetic nano microsphere with high dispersibility and high specific surface, optimizes the preparation process of the nano microsphere, improves the efficiency and specificity of enriching the phosphorylated polypeptide in a complex sample, and meets the requirement of an enrichment method of the large-scale phosphorylated polypeptide.
As shown in fig. 1, an embodiment of the present disclosure provides a method for preparing a hollow core-shell structure nano magnetic microsphere, including:
s100, providing Fe 3 O 4 A nano magnetic microsphere;
s200, fe 3 O 4 Dispersing the nano magnetic microsphere in ethanol by ultrasonic, sequentially adding hexadecyl trimethyl ammonium bromide, tetraethyl silicate and ammonia water, and reacting to obtain Fe with a hollowed-out core-shell structure 3 O 4 @mSiO 2 ；
S300, fe 3 O 4 @mSiO 2 Dispersing in ethanol by ultrasonic wave, adding ammonia water and glycidyloxypropyl trimethoxy silane in sequence to react to obtain Fe 3 O 4 @mSiO 2 @GPS；
S400, fe 3 O 4 @mSiO 2 Dispersing the @ GPS ultrasonic wave in tetrahydrofuran, adding NH 2 -PEG n -NH 2 Reacting to obtain amino modified Fe 3 O 4 @mSiO 2 @NH 2 A nano magnetic microsphere;
s500, fe 3 O 4 @mSiO 2 @NH 2 The nano magnetic microsphere is dispersed in 2, 4-epoxy hexacyclic ring or tetrahydrofuran by ultrasonic, and N, N-carbonyl diimidazole is added for reaction; dispersing the obtained product in alkaline buffer solution, adding N α ,N α -di (carboxymethyl) -L-lysine to give carboxylic acid modified Fe 3 O 4 @mSiO 2 @NTA；
S600, modification of carboxylic acid with Fe 3 O 4 @mSiO 2 Ultrasonic dispersion of @ NTA in ferric trichloride solution to obtain Fe 3 O 4 @mSiO 2 @NTA@Fe 3+ A nano magnetic microsphere.
In some embodiments, in step S100, the Fe 3 O 4 The nanometer magnetic microsphere is prepared from ferric trichloride through a hot solvent reduction method.
Wherein Fe is provided 3 O 4 The nano magnetic microsphere specifically can comprise:
s110, ultrasonically dispersing ferric trichloride in a mixed solution of sodium citrate, anhydrous sodium acetate and ethylene glycol, and heating to dissolve the ferric trichloride;
s120, reacting for 8-16 hours at 180-220 ℃;
s130, sequentially washing the reaction product with water and ethanol for 2-3 times to obtain the magnetic nano Fe with uniform particles 3 O 4 。
In step S110, the amount of ferric trichloride is 0.2-0.3M. In the mixed solution, the mass ratio of the sodium citrate to the anhydrous sodium acetate to the ethylene glycol is 1:60-100:360-450. The mixed solution is heated in an oil bath to dissolve the ferric trichloride.
In step S120, it may be performed in an autoclave. Specifically, the reaction solution obtained in step S110 may be transferred to an autoclave.
In step S130, the amount of water and ethanol used in the washing may be 15-30ml.
In some embodiments, in step S200, fe with a hollowed-out core-shell structure is obtained by reaction 3 O 4 @mSiO 2 The method specifically comprises the following steps:
s210, fe 3 O 4 The nanometer magnetic microspheres are ultrasonically dispersed in ethanol;
s220, sequentially adding cetyl trimethyl ammonium bromide, tetraethyl silicate and ammonia water, and reacting for 2-6 hours;
s230, cleaning the product obtained by the reaction for 2-3 times to obtain Fe with a core-shell structure 3 O 4 @SiO 2 ；
S240, fe 3 O 4 @SiO 2 Ultrasonic dispersing in ethanol or acetone and refluxing for 10-20 hours to obtain Fe with hollow core-shell structure 3 O 4 @mSiO 2 。
In step S210, the ethanol is specifically an aqueous solution of ethanol. Wherein the volume fraction of the ethanol is 50-70%.
In step S220, ammonia may provide an alkaline environment. Specifically, the aqueous ammonia may be added after the tetraethyl silicate is added. Cetyl trimethylammonium bromide, tetraethyl silicate and aqueous ammonia may be added sequentially with mechanical agitation. The mass ratio of the cetyl trimethyl ammonium bromide to the tetraethyl silicate is 1: 1-1:3. The reaction condition is normal temperature.
In step S230, the resultant product is reactedCan be sequentially washed with water and ethanol for 2-3 times respectively to remove Fe with core-shell structure 3 O 4 @SiO 2 Impurities on the surface.
In step S240, the volume of ethanol or acetone may be 50 to 80ml. The number of times of reflux may be one or two, and the duration of each reflux may be 10 hours.
By in step S200, in Fe 3 O 4 When the nano magnetic microsphere, hexadecyl trimethyl ammonium bromide, tetraethyl silicate and ammonia water react, ethanol is adopted as a solvent to be matched with Fe 3 O 4 @SiO 2 Ethanol or acetone is adopted as solvent during the reflux process, and Fe with a hollowed-out core-shell structure shown in figure 3b can be obtained 3 O 4 @mSiO 2 . In the two-step reaction, proper solvent combination is needed to change the ethanol solvent during the reaction or the ethanol or acetone solvent during the reflux, i.e. other solvent combination is needed to be used, the Fe can not be used in the reaction 3 O 4 @SiO 2 The surface of the nano magnetic microsphere forms a hollowed core-shell structure.
In some embodiments, in step S300, the ethanol may be specifically 50 to 80ml of an aqueous ethanol solution. Wherein the volume fraction of the ethanol is 45-75%. The alkaline environment may be provided by ammonia. By dispersing Fe in 3 O 4 @mSiO 2 Adding ammonia water with a final concentration of 0.5-1.5mM into the ethanol water solution. The volume of the glycidyl oxypropyl trimethoxysilane is 15-30ml, and the final concentration is 0.50M. The reaction is mechanically stirred for 2 to 3 hours at room temperature.
Preferably, after the reaction is finished, 20ml of water and ethanol are adopted in sequence, and the mixture is washed once to obtain Fe 3 O 4 @mSiO 2 @GPS。
In some embodiments, in step S400, the resulting amine-modified Fe 3 O 4 @mSiO 2 @NH 2 In the step of nano magnetic microsphere, the NH 2 -PEG n -NH 2 The molar ratio of the catalyst to the tetraethyl silicate is 1:1 to 4:1, the polyethylene glycol polymerization degree n is 2-10, the reaction time is 6-8 hours, and the reaction temperature is 50 ℃. In the step, throughPolyethylene glycol with polymerization degree of 2-10 is selected to obtain amino modified Fe with good dispersion performance 3 O 4 @mSiO 2 @NH 2 A nano magnetic microsphere. Changing the polymerization degree of polyethylene glycol, decreasing or increasing the polymerization degree of polyethylene glycol will lead to the final amino modified Fe 3 O 4 @mSiO 2 @NH 2 The dispersibility of the nano magnetic microsphere is greatly affected, agglomeration can occur, the nano magnetic microsphere cannot be effectively dispersed, and subsequent reaction cannot be carried out, namely, the carboxylic acid modified Fe cannot be obtained 3 O 4 @mSiO 2 NTA and eventually Fe capable of adsorbing trace amounts of phosphorylated polypeptides 3 O 4 @mSiO 2 @NTA@Fe 3+ A nano magnetic microsphere.
Preferably, after the reaction is finished, 20ml of water and ethanol are sequentially adopted, and are respectively washed twice to obtain Fe 3 O 4 @mSiO 2 @GPS。
In some embodiments, in step S500, the concentration of 2, 4-epoxyhexacyclic ring or tetrahydrofuran is 2.5-10mg/ml. The molar ratio of N, N-carbonyl diimidazole to tetraethyl silicate is 1:1 to 4:1, fe 3 O 4 @mSiO 2 @NH 2 The reaction time of the nano magnetic microsphere and N, N-carbonyl diimidazole is 1-2 hours. The reaction temperature was room temperature. After the reaction is completed, 20ml of water is used for washing 2 to 3 times.
The pH of the alkaline buffer solution is 9-11, and the molar ratio of the nα, nα -di (carboxymethyl) -L-lysine to the tetraethyl silicate is 1.2:1-2:1, the Fe 3 O 4 @mSiO 2 @NH 2 The reaction time of the nano magnetic microsphere and the Nalpha, nalpha-di (carboxymethyl) -L-lysine is 4-10 hours, and the reaction temperature is 4 ℃. After the reaction is completed, 20ml of water and ethanol are sequentially adopted to be respectively washed for 2 times to obtain carboxylic acid modified Fe 3 O 4 @mSiO 2 @NTA。
In this step, N, N-carbonyldiimidazole is used as a coupling agent to make Fe 3 O 4 @mSiO 2 @NH 2 -NH of nanomagnetic microsphere surface 2 And N α ,N α -bis (carboxymethyl) -L-lysine (NTA) operatively linked. Changing the kind of coupling agentOr after the dosage, the-NH on the surface of the nano magnetic microsphere can be greatly reduced 2 And N α ,N α Connection success rate of-di (carboxymethyl) -L-lysine (NTA), N cannot be obtained α ,N α -di (carboxymethyl) -L-lysine (NTA) with-NH to the surface of the nanomagnetic microsphere 2 Linking, failure to obtain carboxylic acid modified Fe 3 O 4 @mSiO 2 @NTA。
In some embodiments, in step S600, the ferric trichloride solution is an acetic acid solution of ferric trichloride, and the molar amount of ferric trichloride is 0.8-1.2M. Specifically, fe can be 3 O 4 @mSiO 2 Ultrasonic dispersion of the @ NTA material in 20ml containing 0.8-1.2M FeCl 3 Is reacted in a 0.1M acetic acid solution. Reacting for 5-8 hours at normal temperature. Then the reaction product is washed with 20ml of 0.02mM formic acid solution and pure ethanol solution for 2 to 3 times to obtain Fe 3 O 4 @mSiO 2 @NTA@Fe 3+ A nano magnetic microsphere. The final state of the material is dispersed in 20-80% ethanol aqueous solution.
According to the preparation method of the hollow core-shell structure nano magnetic microsphere, fe with the hollow core-shell structure is prepared 3 O 4 @mSiO 2 The specific surface area of the finally obtained hollow core-shell structure nano magnetic microsphere particles can be remarkably improved, and the loading capacity of the surface functional groups is increased. By connecting silicon dioxide with polyethylene glycol amine radical reagents with different polymerization degrees, fe is improved 3 O 4 Dispersibility of core particles while reducing Fe in acidic environment 3 O 4 Corrosion of the magnetic core; meanwhile, the flexibility of the nano material can be increased by connecting polyethylene glycol with the polymerization degree of 2-10, and the negative effect that the phosphorylated polypeptide is not easy to elute caused by the cavity effect is reduced. By at-NH 2 And N α ,N α When the di (carboxymethyl) -L-lysine (NTA) is connected, N-carbonyl diimidazole is selected as a coupling agent, so that the reaction condition is mild, the reaction time is greatly shortened to 2 hours, and the preparation efficiency of the hollow core-shell structure nano magnetic microsphere is improved.
The preparation of the hollow core-shell structure nano magnetic microsphere is described in detail below by referring to fig. 2 through a specific embodiment.
Example 1 preparation of hollow core-shell Structure nanomagnetic microspheres
1. Ultrasonically dispersing 0.2M ferric trichloride into 40ml of mixed solution of sodium citrate, anhydrous sodium acetate and ethylene glycol, wherein the mass ratio of three substances in the mixed solution is 1:80:400, heating and dissolving the mixed solution in an oil bath, then, putting the reaction solution into an autoclave, reacting for 10 hours at 200 ℃, after the reaction is finished, washing the product with 20ml of water and ethanol for 3 times in sequence to obtain Fe with uniform particles 3 O 4 A nano magnetic microsphere. Fe obtained 3 O 4 An electron microscope image of the nano magnetic microsphere is shown in fig. 3 a.
2. Fe obtained in step 1 3 O 4 Dispersing in 60ml of aqueous solution containing 60% ethanol by ultrasonic, sequentially adding cetyl trimethyl ammonium bromide, tetraethyl silicate and ammonia water under the condition of mechanical stirring, wherein the mass ratio of the cetyl trimethyl ammonium bromide to the tetraethyl silicate is 1:2, reacting for 4 hours at normal temperature, and respectively washing with 20ml of water and ethanol for 2 times after the reaction is finished to obtain Fe with a core-shell structure 3 O 4 @SiO 2 。Fe 3 O 4 @SiO 2 Dispersing in 60ml acetone solution, heating and refluxing for 10 hours, and repeating the refluxing process for 3 times to obtain the Fe with the hollowed-out core-shell structure 3 O 4 @mSiO 2 . Fe of the obtained hollowed-out core-shell structure 3 O 4 @mSiO 2 The electron microscope of (a) is shown in figure 3 b.
3. Fe obtained in step 2 3 O 4 @mSiO 2 Dispersing with ultrasound in 60ml 67% ethanol water solution, adding ammonia water with final concentration of 1mM, adding 20ml ethanol solution containing glycidyloxypropyl trimethoxysilane (GPS, 0.50M) dropwise, mechanically stirring at room temperature for 2 hr, washing the product with 20ml water and ethanol sequentially to obtain Fe 3 O 4 @mSiO 2 @ GPS material.
4. Fe obtained in step 3 3 O 4 @mSiO 2 Ultrasound dispersion of @ GPS in NH containing 2 -PEG 2 -NH 2 Wherein NH is in tetrahydrofuran solution 2 -PEG 2 -NH 2 The molar ratio of the tetraethyl silicate in the step 2 is 2:1, the polymerization degree of polyethylene glycol is 2, the dispersion solution is reacted for 6 hours at 50 ℃, after the temperature of the reaction solution is reduced to room temperature, 20ml of water and ethanol are used for 2 times to obtain amino modified Fe 3 O 4 @mSiO 2 @NH 2 A nano magnetic microsphere. The obtained amino-modified Fe 3 O 4 @mSiO 2 @NH 2 An electron microscope image of the nano magnetic microsphere is shown in fig. 3 c.
5. Fe obtained in step 4 3 O 4 @mSiO 2 @NH 2 The microspheres are dispersed in 2, 4-epoxy hexacyclic ring, the concentration of the solution is 5mg/ml, N, N-carbonyl diimidazole is added, and the molar ratio of the N, N-carbonyl diimidazole to the tetraethyl silicate in the step 2 is 2:1, after reacting for 2 hours at room temperature, washing with 20ml of water for 2 times; dispersing the above product in alkaline buffer solution of pH 10, adding appropriate amount of N α ,N α -di (carboxymethyl) -L-lysine, N α ,N α -molar ratio of di (carboxymethyl) -L-lysine to tetraethyl silicate in step 2 1.5:1, reacting for 8 hours, washing with 20ml of water and ethanol 2 times each to obtain carboxylic acid modified Fe 3 O 4 @mSiO 2 @NTA material.
6. Fe obtained in step 5 3 O 4 @mSiO 2 The @ NTA material was ultrasonically dispersed in 60ml of a solution containing 1M FeCl 3 After reacting for 6 hours at normal temperature, the material is washed with 20ml of 20mM formic acid solution and pure ethanol solution for 2 times in sequence to obtain the final Fe 3 O 4 @mSiO 2 @NTA@Fe 3+ The nanometer magnetic microsphere is finally dispersed and stored in 60% concentration alcohol water solution.
Comparative example 1
The difference from example 1 is that Fe in step 2 3 O 4 @SiO 2 The dispersed solvent acetone is replaced by diethyl ether; and does not include steps 3-6.
The product prepared in step 2 of comparative example 1 was subjected to electron microscopy scanning, respectively, for example 1. Wherein the results obtained in example 1 are shown in FIG. 3b, and the results obtained in comparative example 1 are shown in FIG. 6 a. Diethyl ether is adopted as Fe 3 O 4 @SiO 2 The result obtained by the dispersion solvent of (2) is that acetone is adopted as Fe 3 O 4 @SiO 2 For a comparison of the results obtained with the dispersion solvent of (a), refer to fig. 7. Thus, fe in step 2 of example 1 was changed 3 O 4 @SiO 2 Dispersed solvent acetone can not obtain Fe with hollowed-out core-shell structure 3 O 4 @mSiO 2 。
It should be noted that the foregoing describes some embodiments of the present disclosure. Other embodiments are within the scope of the following claims. In some cases, the actions or steps recited in the claims may be performed in a different order than in the embodiments described above and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.
Based on the same inventive concept, the embodiment of the disclosure also provides a hollow core-shell structure nano magnetic microsphere corresponding to the method of any embodiment. The hollow core-shell structure nano magnetic microsphere is prepared by the preparation method according to the technical scheme. The electron microscope image is shown in fig. 3 d. The near infrared characterization result is shown in figure 4.
The disclosure provides a preparation method of the hollow core-shell structure nano magnetic microsphere according to any one of the technical schemes or application of the hollow core-shell structure nano magnetic microsphere prepared by the preparation method in specific enrichment or purification of trace phosphorylated polypeptides.
By means of specific embodiments, the preparation of the hollow-core-shell structure nano magnetic microsphere and the enrichment process of the phosphorylated polypeptide are described in detail.
EXAMPLE 2 purification of phosphorylated Polypeptides in beta-case Using hollow-core Shell nanomagnetic microspheres
1. 1mg of beta-casein is dissolved in 1ml of 50mM ammonium bicarbonate buffer solution to obtain a beta-casein solution with the concentration of 1 mg/ml;
2. taking 10ug of beta-casein, adding pancreatic protein according to the proportion of 1:50, performing enzyme digestion for 12 hours under the water bath condition of 37 ℃, adding trifluoroacetic acid with the final concentration of 1% after enzyme digestion is finished to terminate enzyme digestion reaction, and obtaining beta-casein enzyme digestion peptide fragments after vacuum drying;
3. fe prepared in example 1 was dissolved in 100ul of binding buffer (1% trifluoroacetic acid, 80% acetonitrile) 3 O 4 @mSiO 2 @NTA@Fe 3+ Washing for three times for standby;
4. dissolving the polypeptide obtained in step 2 in 100ul of binding buffer, and transferring to a buffer containing balanced Fe 3 O 4 @mSiO 2 @NTA@Fe 3+ Uniformly dispersing the magnetic microspheres in a binding buffer solution in a magnetic microsphere centrifuge tube, binding for 30 minutes, and uniformly dispersing the settled magnetic microspheres again every 10 minutes;
5. after the binding is completed, the magnetic microspheres are washed three times with 100ul of binding buffer solution, and washed twice with 100ul of deionized water;
6. the phosphorylated peptides bound to the magnetic microspheres were eluted with 100ul of 1% and 2.5% ammonia water, respectively, and the two eluents were combined, and after rapid vacuum spin-drying, the polypeptides before and after purification were detected by LC-MS/MS after re-dissolution with 0.1% formic acid water, respectively, and the purification effect was evaluated, as shown in fig. 5.
As can be seen from fig. 5, after the phosphopeptide enrichment is performed on the β -casein protein by using the hollow core-shell structure nano magnetic microsphere prepared in example 1, the phosphorylated protein in more abundance regions can be detected by LC-MS/MS. Therefore, the hollow core-shell structure nano magnetic microsphere prepared in the embodiment 1 can be used as an affinity reagent for carrying out specific enrichment and purification on trace phosphorylated polypeptides in complex biological samples, and has excellent specific enrichment and purification effects.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined under the idea of the present disclosure, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in details for the sake of brevity.
Furthermore, it will be apparent to one skilled in the art that, in order to simplify the description and discussion, and in order not to obscure the embodiments of the disclosure, specific details are set forth to describe example embodiments of the disclosure, that the embodiments of the disclosure may be practiced without, or with variations in, these specific details. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.
While the present disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of those embodiments will be apparent to those skilled in the art in light of the foregoing description.
The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Accordingly, any omissions, modifications, equivalents, improvements, and the like, which are within the spirit and principles of the embodiments of the disclosure, are intended to be included within the scope of the disclosure.
Claims (7)
1. The preparation method of the hollow core-shell structure nano magnetic microsphere is characterized by comprising the following steps of:
providing Fe 3 O 4 A nano magnetic microsphere;
fe is added to 3 O 4 Nanometer magnetic microsphere, hexadecyl trimethyl ammonium bromide and tetraethyl silicate react in ethanol in alkaline environment to obtain Fe with hollowed-out core-shell structure 3 O 4 @mSiO 2 ；
Fe is added to 3 O 4 @mSiO 2 Dispersing in ethanol by ultrasonic wave, adding glycidyl oxypropyl trimethoxy silane, reacting in alkaline environment, and obtaining Fe at normal temperature 3 O 4 @mSiO 2 @GPS；
Fe is added to 3 O 4 @mSiO 2 Dispersing the @ GPS ultrasonic wave in tetrahydrofuran, adding NH 2 -PEG n -NH 2 Reacting to obtain amino modified Fe 3 O 4 @mSiO 2 @NH 2 A nano magnetic microsphere;
fe is added to 3 O 4 @mSiO 2 @NH 2 The nano magnetic microsphere is dispersed in 2, 4-epoxy hexacyclic ring or tetrahydrofuran by ultrasonic, and N, N-carbonyl diimidazole is added for reaction; dispersing the obtained product in alkaline buffer solution, adding N α ,N α -di (carboxymethyl) -L-lysine to give carboxylic acid modified Fe 3 O 4 @mSiO 2 @NTA；
Modification of carboxylic acid with Fe 3 O 4 @mSiO 2 Ultrasonic dispersion of @ NTA in ferric trichloride solution to obtain Fe 3 O 4 @mSiO 2 @NTA@Fe 3+ A nano magnetic microsphere;
the reaction is carried out to obtain Fe with a hollowed-out core-shell structure 3 O 4 @mSiO 2 The method specifically comprises the following steps:
fe is added to 3 O 4 The nanometer magnetic microspheres are ultrasonically dispersed in ethanol;
sequentially adding cetyl trimethyl ammonium bromide, tetraethyl silicate and ammonia water, and reacting for 2-6 hours; respectively cleaning the products obtained by the reaction for 2-3 times by adopting water and ethanol in turn to obtain Fe with a core-shell structure 3 O 4 @SiO 2 ；
Fe is added to 3 O 4 @SiO 2 Ultrasonic dispersing in ethanol or acetone and refluxing for 10-20 hours to obtain Fe with hollow core-shell structure 3 O 4 @mSiO 2 The method comprises the steps of carrying out a first treatment on the surface of the The mass ratio of the cetyl trimethyl ammonium bromide to the tetraethyl silicate is 1: 1-1:3;
the obtained Fe 3 O 4 @mSiO 2 In the step of @ GPS, the volume of the ethanol is 50-80 ml; the alkaline environment is provided for ammonia water, the final concentration of the ammonia water is 0.5-1.5mM, the volume of the glycidyl oxypropyl trimethoxysilane is 15-30ml, and the final concentration is 0.50M;
the Fe modified by the obtained amino group 3 O 4 @mSiO 2 @NH 2 In the step of nano magnetic microsphere, the NH 2 -PEG n -NH 2 The molar ratio of the catalyst to the tetraethyl silicate is 1:1 to 4:1, the polymerization degree n of the ethylene glycol is 2-10, the reaction time is 6-8 hours, and the reaction temperature is 50 ℃.
2. The method according to claim 1, wherein the Fe 3 O 4 The nano magnetic microsphere is prepared from 0.2-0.3M ferric trichloride by a hot solvent reduction method.
3. The method of claim 1, wherein the carboxylic acid-modified Fe is obtained 3 O 4 @mSiO 2 In the step @ NTA, the molar ratio of N, N-carbonyldiimidazole to tetraethyl silicate is 1:1 to 4:1, fe 3 O 4 @mSiO 2 @NH 2 The reaction time of the nano magnetic microsphere and N, N-carbonyl diimidazole is 1-2 hours.
4. The method of claim 1, wherein the carboxylic acid-modified Fe is obtained 3 O 4 @mSiO 2 In the step @ NTA, the pH of the alkaline buffer solution is 9-11, and the pH of the alkaline buffer solution is N α ,N α -molar ratio of di (carboxymethyl) -L-lysine to the tetraethyl silicate 1.2:1-2:1, the Fe 3 O 4 @mSiO 2 @NH 2 Nanometer magnetic microsphere and N α ,N α The reaction time of the di (carboxymethyl) -L-lysine is 4-10 hours, and the reaction temperature is 4 ℃.
5. The method according to claim 1, wherein Fe is obtained 3 O 4 @mSiO 2 @NTA@Fe 3+ In the step of the nano magnetic microsphere, the ferric trichloride solution is acetic acid solution of ferric trichloride, the molar quantity of the ferric trichloride is 0.8-1.2M, and the reaction time is 5-8 h.
6. A hollow core-shell structure nano-magnetic microsphere, characterized in that the hollow core-shell structure nano-magnetic microsphere is prepared by the preparation method according to any one of claims 1 to 5.
7. Use of the hollow core-shell structured nano-magnetic microsphere of claim 6 in the specific enrichment or purification of trace amounts of phosphorylated polypeptides.
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