CN101434641A - Zirconium phosphoester nano magnetic beads, as well as preparation and application thereof - Google Patents
Zirconium phosphoester nano magnetic beads, as well as preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a phosphate zirconium nano magnetic particle with the structural representation on the right, and the preparation and application thereof; the preparation method comprises the steps that phosphate groups are induced to the surface of the nano magnetic particle which is wrapped with silicon dioxide on the surface and takes ferriferrous oxide as a magnetic inner core; then, a phosphate zirconium layer is formed on the surface by the mutual action between zirconium ions and the phosphate groups on the surface, thereby successively preparing a nano magnetic particle having high efficiency, high-selectivity separation and enrichment feature to peptide phosphate. The nano magnetic particle is mainly used for separating the enriched peptide phosphate from biological samples, with high efficiency and under the action of a magnetic field. The extracted peptide phosphate can be directly analyzed and represented by mass spectrum.
Description
Technical field
The present invention relates to the separation and concentration of phosphorylated peptide, specifically a kind of Zirconium phosphoester nano magnetic beads and preparation thereof, with and application in the selective separation enrichment of phosphorylated peptide.
Background technology
Proteinic compartment analysis is the research focus of bioseparation analysis field in recent years, proteinic posttranslational modification is the important factor that influences protein structure and function, in the regulation and control of bio-chemical pathway, play an important role, and wherein protein phosphorylation be the most common also be most important a kind of posttranslational modification mode, be the focus and the difficult point of proteomics research.Protein phosphorylation and dephosphorylation be the whole process of attemperator's vital movement almost, comprises the propagation of cell, grows and differentiation, nervous activity, Muscle contraction, metabolism, main signal transfer mode known to tumour generation etc., protein phosphorylation are still present.
The evaluation of phosphorylated protein can be finished detection with MALDI-TOF MS on the basis of enzymolysis, but the ion signal intensity of phosphated peptide section tends to be restrained by the ion signal of residual non-phosphorylating peptide section behind the enzymolysis, and the concentration of phosphorylated protein in actual sample is very low again, so phosphated peptide section is carried out pre-separation and pre-concentration is a kind of eliminating non-phosphorylating peptide mass spectroscopy interferential effective ways.The separation of phosphorylated peptide and enrichment comparatively effective means are immobilization metal chelating affinity chromatographies, the cardinal principle of this method be utilize phosphated peptide section with phosphate radical and the immobilization metal affiliation carrier on the interaction of highly selective takes place between immobilized metal ion, but not then than difficulty special highly selective taking place with it, phosphated peptide section interacts, Gu this can reach the selective separation of phosphated peptide section and the effect of enrichment.
Recently, metal zirconium or titanium ion is immobilized in being that the chromatographic stationary phase surface of matrix and the analysis that is applied to phosphorylated peptide have successfully report, TiO recently with silica gel
2And ZrO
2Microballoon, Fe
3O
4/ TiO
2Shell hole nano particle etc. also is used to purifying and enrichment acid peptide (document 1.Yu-Chie Chen et.al " Fe3O4/TiO2 Core/Shell Nanoparticles as AffinityProbes for the Analysis of Phosphopeptides Using TiO2 Surface-AssistedLaser Desorption/Ionization Mass Spectrometry " " AnalyticalChemistry ", 2005,77,5912-5919.).But with immobilized surface and the method that is applied to the separation and concentration of the phosphorylated peptide announcement that then do not appear in the newspapers in the functionalized nano magnetic bead of metal zirconium ion.Nanometer magnetic bead has the dual nature of nanoscale and super paramagnetic, in recent years received increasing concern, field (document 2.Milan J.Beneset.al " Preparation and properties of magnetic nano-and microsized particles forbiological and environmental separations " such as chemistry, biology, medical science, environment have been widely used in, " Journal of SeparationScience ", 2007,30,1751-1772.).
Summary of the invention
The object of the present invention is to provide a kind of Zirconium phosphoester nano magnetic beads and preparation thereof and application, it can finish the efficient highly selective separation and concentration of phosphorylated peptide from the biological sample of complexity quickly and easily under the effect in magnetic field.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of Zirconium phosphoester nano magnetic beads is characterized in that: the structural representation of Zirconium phosphoester functionalized nano magnetic bead is as follows,
The magnetic bead kernel of described Zirconium phosphoester nano magnetic beads is a ferroferric oxide magnetic nanoparticle, and the magnetic bead particles mean diameter is (10nm).
Can operate as follows,
1. by the strong avidity in ferroferric oxide nano granules surface, ferriferrous oxide nano magnetic bead particles surface coverage last layer silicon-dioxide;
Be specially: get 0.15-0.35g ferriferrous oxide nano magnetic bead and under ultrasonication, be dispersed in the ethanol solution of 30-120ml, under 25-55 ℃ of stirring in water bath, add 1-5ml tetrasilicic acid ethyl ester, react after 1-5 hour, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead with absolute ethanol washing after, the dehydrated alcohol that adds 10-120ml again, in 45-75 ℃ of water-bath backflow 5-20 hour; Hold magnetic bead with magnet, remove supernatant liquor; Repeat above-mentioned steps once.
2. coating is gone up the nanometer magnetic bead of silicon dioxide layer by amino group on the amination chemical reaction bonding;
Be specially: in the covering that will prepare the nanometer magnetic bead of silicon-dioxide in vacuum drier 20-100 ℃ place after 2-24 hour, nanometer magnetic bead after the drying is put into the 20-50ml anhydrous toluene solution, in toluene solution, add the 1-3ml3-aminopropyl triethoxysilane, under N2 protection 90-120 ℃ stirring and refluxing 4-15 hour, hold magnetic bead with magnet then, remove supernatant liquor, obtain amidized nanometer magnetic bead, use toluene successively, distilled water and absolute ethanol washing were placed 5-15 hour in 25-60 ℃ vacuum drier then;
3. nanometer magnetic bead after the amination and POCl
3Reaction obtains the phosphorylation nanometer magnetic bead.
Be specially: the amidized nanometer magnetic bead that will prepare places and comprises 20-80mM POCl
3, the 20-50mM 2 20-80mL anhydrous acetonitrile organic solution in the reaction 4-18 hour; Hold magnetic bead with magnet, remove supernatant liquor, magnetic bead is used anhydrous acetonitrile and distilled water wash successively;
With the zirconium ion chelating in phosphorylation nanometer magnetic bead surface, the affine magnetic nanoparticle of being fixed metal-chelating;
Be specially: the 30-60mL ZrOCl that then nanometer magnetic bead of phosphate group on the bonding is placed 10-50mM
2Reaction is 8-24 hour in the aqueous solution, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead is aqueous acetic acid, the distilled water wash of 10-15% with the volume content of 100-200mM sodium-chlor successively, in 20-80 ℃ vacuum drier, placed 5-15 hour then, obtain Zirconium phosphoester nano magnetic beads.
Described Zirconium phosphoester nano magnetic beads can be used for the enrichment and the purifying of phosphorylated peptide in the protein zymolyte.Promptly under the proper acidic condition, be used for the separation and concentration of highly selective of the phosphorylated peptide of protein sample, remove non-phosphorylating peptide section by washing, avoid the interference of non-phosphorylating peptide in the mass spectroscopy.
The present invention has following advantage:
1. covering the nanometer magnetic bead of going up silicon-dioxide can effectively protect Z 250 magnetic nuclear by the erosion of bronsted lowry acids and bases bronsted lowry; and can further carry out reaction kinetic at silica sphere, the nanometer magnetic bead of key and last phosphoric acid ester can stop Z 250 magnetic to examine non-specific property absorption non-phosphopeptide effectively.
2. the characteristic bond zirconium ion of the high-specific surface area of nanometer magnetic bead and super paramagnetic prepares the nanometer magnetic bead of gained to the affine interaction principle of the highly selective of phosphorylated peptide, the efficiently separation and concentration of phosphorylated peptide can be under the effect in magnetic field, finished quickly and easily, the sample loss that waits pre-treatment step to bring because of centrifugal can be reduced from the biological sample of complexity.
3. because the material of nanoscale has very big specific surface area, so nanometer magnetic bead can key and last more Zirconium phosphoester functional group, thereby Zirconium phosphoester nano magnetic beads has accumulation ability more efficiently.
4. Zirconium phosphoester nano magnetic beads has wetting ability preferably, can be dissolved in well in the aqueous solution, and good biocompatibility and stability are arranged under physiological condition, and can interact with the phosphorylated peptide highly selective in the protein sample.
The present invention at first phosphate group is introduced that the surface is coated with silicon oxide, be the nano magnetic particle surface of magnetic kernel with the Z 250, interaction by metal zirconium ion and surface phosphoric acid group forms surface phosphoric acid ester zirconium layer then, has successfully prepared the magnetic nanoparticle of a kind of, highly selective separation efficient to the phosphorylated peptide tool and enriched character.This nanometer magnetic bead is mainly used under the action of a magnetic field high efficiency separation enriching phosphated peptide from biological sample, and the phosphorylated peptide that is extracted can directly adopt mass spectrum to carry out analysis and characterization.
Description of drawings
Fig. 1 is the preparation synoptic diagram of Zirconium phosphoester nano magnetic beads;
Fig. 2 is that Zirconium phosphoester nano magnetic beads is to the enrichment of phosphorylated peptide in the phosphorylated protein alpha-casein enzymolysis product and the MALDI-TOF mass spectrum of purifying; The sequence of phosphorylated peptide sees Table 1;
Fig. 3 is that Zirconium phosphoester nano magnetic beads is to phosphorylated peptide in the phosphorylated protein beta-casein enzymolysis product and the enrichment of standard phosphorylated tyrosine peptide section and the MALDI-TOF mass spectrum of purifying; The sequence of phosphorylated peptide sees Table 2.
Embodiment
Introduce the present invention with specific embodiment below.
Embodiment 1 utilizes Zirconium phosphoester nano magnetic beads to realize the enrichment of phosphorylated peptide
The preparation of Zirconium phosphoester nano magnetic beads:
1. Z 250 kernel magnetic bead is dispersed in the ethanol solution of 30ml under ultrasonication, under 40 ℃ of water-bath and stirring, add 1.0ml tetrasilicic acid ethyl ester (TEOS), react after 2 hours, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead with absolute ethanol washing after, add the dehydrated alcohol of 80ml again, in 60 ℃ of water-baths, refluxed 12 hours, hold magnetic bead with magnet, remove supernatant liquor.
2. after the nanometer magnetic bead of the last silicon-dioxide of covering is placed 12 hours in vacuum drier, in the 35ml anhydrous toluene solution, add 1.5ml3-aminopropyl triethoxysilane (APTES), N2 protection and 110 ℃ of following stirring and refluxing 10 hours, hold magnetic bead with magnet then, remove supernatant liquor, obtain amidized nanometer magnetic bead, use toluene successively, distilled water and absolute ethanol washing were placed 12 hours in 25 ℃ vacuum drier then; Repeat above-mentioned steps once.
3. amidized nanometer magnetic bead places and comprises 40mM POCl
3, reaction is 12 hours in the 40mL anhydrous acetonitrile organic solution of 40mM 2, holds magnetic bead with magnet, removes supernatant liquor, and magnetic bead is used anhydrous acetonitrile and distilled water wash successively.
4. the nanometer magnetic bead of phosphate group places the 50ml ZrOCl of 30mM on the bonding
2Reaction is 12 hours in the aqueous solution, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead is 10% aqueous acetic acid, distilled water wash successively with the volume content of 200mM sodium-chlor, in 30 ℃ vacuum drier, placed 12 hours then, obtain Zirconium phosphoester nano magnetic beads, obtain Zirconium phosphoester nano magnetic beads.
The alpha-casein of the preparation of sample solution: 1mg and beta-casein be dissolved in 1mL respectively, in the Ammonium bicarbonate food grade solution of 50mM (pH 8.2), add trypsinase according to the ratio with tryptic mass ratio 40:1 and carry out enzyme digestion reaction, the reaction times is 16h, and hydrolysis temperature is controlled at 37 ℃.It is standby that the proteolysis solution that obtains places-30 ℃ of refrigerators to preserve.
The enrichment of phosphorylated peptide and maldi analysis: Zirconium phosphoester nano magnetic beads is dispersed in the anhydrous second eyeball solution.The enzymolysis solution of above-mentioned phosphorylated protein alpha-casein and beta-casein is dissolved in 50%ACN respectively, in the 5.0%HAC aqueous solution, get sample solution and 10u1 immobilization zirconium metal ion chela and the affinity chromatography particle solution of 2 μ L then, hatch 10-60min, hold Zirconium phosphoester nano magnetic beads with magnet, remove supernatant liquor, then successively with containing 10 μ L, 50% ACN, 200mM NaCl, the mixing solutions of 5.0%HAC and 10 μ L, 50% ACN, the mixing solutions of 5.0% HAC cleans 5-20min, then with holding immobilization zirconium metal ion chela and affinity chromatography particle with magnet behind the ultrasonic wash-out 10-60min of the ammoniacal liquor of 5-20%, get supernatant liquor and put into centrifuge tube, lyophilize, 5 μ L contain 1% H
3PO
4DHB (10-50mg/mL) solution join in the dry centrifuge tube of crossing, get 0.5 μ L solution deposition on the MALDI target, form cocrystallization, carry out the MALDI-TOF mass spectroscopy.
Analytical results: as seen by Fig. 2 and Fig. 3, the phosphorylated peptide that comes from phosphorylated protein alpha-casein and the beta-casein enzymolysis product is caught by Zirconium phosphoester nano magnetic beads, but not phosphorylated peptide then can be illustrated special enrichment and the separation and purification phosphorylated peptide of nanometer magnetic bead energy that Zirconium phosphoester is modified by easy wash-out.
Table 1. is detected phosphorylated peptide in the alpha-casein enzymolysis solution
| Sequence number | [M+H] + | The phosphorylation site number | Aminoacid sequence |
| α1 | 1237.50 | 1 | TVDME[ PS]TEVF |
| α2 | 1254.52 | 1 | TVD[ Mo]ME[ PS]TEVF |
| α3 | 1466.61 | 1 | TVDME[ PS]TEVFTK |
| α4 | 1482.61 | 1 | TVD[ Mo]E[ PS]TEVFTK |
| α5 | 1538.59 | 2 | EQL[ PS］T[ PS]EENSKK |
| α6 | 1660.79 | 1 | VPQLEIVPN[ PS]AEER |
| α7 | 1832.83 | 1 | YLGEYLIVPN[ PS]AEER |
| α8 | 1927.69 | 2 | DIG[ PS]E[ PS]TEDQAMEDIK |
| α9 | 1951.95 | 1 | YKVPQLEIVPN[ PS]AEER |
| α10 | 2079.04 | 1 | KKYKVPQLEIVPN[ PS]AEERL |
| α11 | 2619.04 | 4 | NTMEHV[ PS］[ PS］[ PS]EESII[ PS]QET YK |
| α12 | 2720.91 | 5 | QMEAE[ PS]I[ PS][ PS][ PS]EEIVPNPN [ PS]VEQK |
| α13 | 2935.15 | 3 | KEKVNEL[ PS]KDIG[ PS]E[ PS]TEDQ AMEDIKQ |
| α14 | 3008.01 | 4 | NANEEEYSIG[ PS][ PS][ PS]EE[ PS]AE VATEEVK |
| α15 | 3087.99 | 5 | NANEEEY[ PS］IG[ PS][ PS］[ PS]EE[ PS] AEVATEEVK |
Table 2. is detected phosphorylated peptide in the beta-casein enzymolysis solution
Embodiment 2
The preparation of Zirconium phosphoester nano magnetic beads:
1.0.15g Z 250 kernel magnetic bead is dispersed in the ethanol solution of 60ml under ultrasonication, under 45 ℃ of water-bath and stirring, add 1.5ml tetrasilicic acid ethyl ester (TEOS), react after 3 hours, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead with absolute ethanol washing after, add the dehydrated alcohol of 90ml again, in 45 ℃ of water-baths, refluxed 13 hours, hold magnetic bead with magnet, remove supernatant liquor.
2. after the nanometer magnetic bead of the last silicon-dioxide of covering is placed 8 hours in vacuum drier, in the 20ml anhydrous toluene solution, add 1.5ml 3-aminopropyl triethoxysilane (APTES), at N
2Protection and 90 ℃ of following stirring and refluxing 4 hours hold magnetic bead with magnet then, remove supernatant liquor, obtain amidized nanometer magnetic bead, use toluene successively, distilled water and absolute ethanol washing, placement 6 hours in 30 ℃ vacuum drier then; Repeat above-mentioned steps once.
3. amidized nanometer magnetic bead places and comprises 30mM POCl
3, reaction is 8 hours in the 30mL anhydrous acetonitrile organic solution of 30mM 2, holds magnetic bead with magnet, removes supernatant liquor, and magnetic bead is used anhydrous acetonitrile and distilled water wash successively.
4. the nanometer magnetic bead of phosphate group places the 30ml ZrOCl of 20mM on the bonding
2Reaction is 12 hours in the aqueous solution, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead is 11% aqueous acetic acid, distilled water wash successively with the volume content of 100mM sodium-chlor, in 20 ℃ vacuum drier, placed 6 hours then, obtain Zirconium phosphoester nano magnetic beads.Obtain Zirconium phosphoester nano magnetic beads.
Embodiment 3
The preparation of Zirconium phosphoester nano magnetic beads:
1.0.25g Z 250 kernel magnetic bead is dispersed in the ethanol solution of 80ml under ultrasonication, under 50 ℃ of water-bath and stirring, add 2.0ml tetrasilicic acid ethyl ester (TEOS), react after 4 hours, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead with absolute ethanol washing after, add the dehydrated alcohol of 100ml again, in 65 ℃ of water-baths, refluxed 14 hours, hold magnetic bead with magnet, remove supernatant liquor.
2. after the nanometer magnetic bead of the last silicon-dioxide of covering is placed 16 hours in vacuum drier, in the 40ml anhydrous toluene solution, add 1.5ml 3-aminopropyl triethoxysilane (APTES), at N
2Protection and 115 ℃ of following stirring and refluxing 8 hours hold magnetic bead with magnet then, remove supernatant liquor, obtain amidized nanometer magnetic bead, use toluene successively, distilled water and absolute ethanol washing, placement 10 hours in 35 ℃ vacuum drier then; Repeat above-mentioned steps once.
3. amidized nanometer magnetic bead places and comprises 60mM POCl
3, reaction is 16 hours in the 60mL anhydrous acetonitrile organic solution of 35mM 2, holds magnetic bead with magnet, removes supernatant liquor, and magnetic bead is used anhydrous acetonitrile and distilled water wash successively;
4. the nanometer magnetic bead of phosphate group places the 40ml ZrOCl of 35mM on the bonding
2Reaction is 12 hours in the aqueous solution, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead is 12% aqueous acetic acid, distilled water wash successively with the volume content of 150mM sodium-chlor, in 40 ℃ vacuum drier, placed 10 hours then, obtain Zirconium phosphoester nano magnetic beads.Obtain Zirconium phosphoester nano magnetic beads.
Embodiment 4
The preparation of Zirconium phosphoester nano magnetic beads:
1.0.25g Z 250 kernel magnetic bead is dispersed in the ethanol solution of 100ml under ultrasonication, under 30 ℃ of water-bath and stirring, add 2.5ml tetrasilicic acid ethyl ester (TEOS), react after 5 hours, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead with absolute ethanol washing after, add the dehydrated alcohol of 120ml again, in 70 ℃ of water-baths, refluxed 15 hours, hold magnetic bead with magnet, remove supernatant liquor.
2. after the nanometer magnetic bead of the last silicon-dioxide of covering is placed 20 hours in vacuum drier, in the 50ml anhydrous toluene solution, add 1.5ml 3-aminopropyl triethoxysilane (APTES), at N
2Protection and 120 ℃ of following stirring and refluxing 12 hours hold magnetic bead with magnet then, remove supernatant liquor, obtain amidized nanometer magnetic bead, use toluene successively, distilled water and absolute ethanol washing, placement 14 hours in 40 ℃ vacuum drier then; Repeat above-mentioned steps once.；
3. amidized nanometer magnetic bead places and comprises 70mM POCl
3, reaction is 18 hours in the 70mL anhydrous acetonitrile organic solution of 50mM 2, holds magnetic bead with magnet, removes supernatant liquor, and magnetic bead is used anhydrous acetonitrile and distilled water wash successively.
4. the nanometer magnetic bead of phosphate group places the 60ml ZrOCl of 40mM on the bonding
2Reaction is 12 hours in the aqueous solution, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead is 15% aqueous acetic acid, distilled water wash successively with the volume content of 180mM sodium-chlor, in 60 ℃ vacuum drier, placed 14 hours then, obtain Zirconium phosphoester nano magnetic beads.Obtain Zirconium phosphoester nano magnetic beads.
Magnetic bead
SEQUENCE?LISTING
＜110〉Dalian Inst of Chemicophysics, Chinese Academy of Sciences
＜120〉a kind of Zirconium phosphoester nano magnetic beads and preparation thereof and application
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Claims (3)
1. Zirconium phosphoester nano magnetic beads, it is characterized in that: the structural representation of Zirconium phosphoester functionalized nano magnetic bead is as follows,
2. the preparation method of the described Zirconium phosphoester nano magnetic beads of claim 1 is characterized in that: can operate as follows,
(1) the nanometer magnetic bead surface coats layer of silicon dioxide: get 0.15-0.25g ferriferrous oxide nano magnetic bead and be dispersed in the ethanol solution of 30-120ml under ultrasonication, under 25-55 ℃ of stirring in water bath, add 1-5ml tetrasilicic acid ethyl ester, react after 1-5 hour, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead with absolute ethanol washing after, the dehydrated alcohol that adds 10-120ml again, in 45-75 ℃ of water-bath backflow 5-20 hour; Hold magnetic bead with magnet, remove supernatant liquor; Holding magnetic bead with gained magnet is that raw material can repeat above-mentioned steps once again;
(2) nanometer magnetic bead of silicon-dioxide carries out ammoxidation in the coating: with the nanometer magnetic bead of silicon-dioxide in the covering of step (1) preparation in vacuum drier 20-100 ℃ place 2-24 hour after, nanometer magnetic bead after the drying is put into the 10-50ml anhydrous toluene solution, in toluene solution, add the 1-5ml3-aminopropyl triethoxysilane, at N
2Protect following 90-120 ℃ of stirring and refluxing 5-15 hour, hold magnetic bead with magnet then, remove supernatant liquor, obtain amidized nanometer magnetic bead, use toluene successively, distilled water and absolute ethanol washing were placed 5-15 hour in 40-80 ℃ vacuum drier then;
(3) nanometer magnetic bead after the amination and POCl
3Reaction obtains the phosphorylation nanometer magnetic bead: the amidized nanometer magnetic bead of step (2) preparation is placed comprise 20-80mM POCl
3, 20-50mM2,4, in the 20-80mL anhydrous acetonitrile organic solution of 6-trimethylpyridine the reaction 4-18 hour; Hold magnetic bead with magnet, remove supernatant liquor, magnetic bead is used anhydrous acetonitrile and distilled water wash successively;
(4) nanometer magnetic bead chela after the amination and last functional group Zirconium phosphoester: place the 30-60mL ZrOCl2 aqueous solution of 10-50mM to react 8-15 hour the nanometer magnetic bead of phosphate group on the bonding of step (3) preparation, hold magnetic bead with magnet, remove supernatant liquor, magnetic bead is used aqueous acetic acid, the distilled water wash of the volume content 10-15% of 100-200mM sodium-chlor successively, in 20-80 ℃ vacuum drier, placed 5-15 hour then, obtain Zirconium phosphoester nano magnetic beads.
3. the application of the described Zirconium phosphoester nano magnetic beads of claim 1 is characterized in that: described Zirconium phosphoester nano magnetic beads is used for the enrichment and the purifying of protein zymolyte phosphorylated peptide.
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| CN102350326A (en) * | 2011-07-19 | 2012-02-15 | 武汉大学 | Preparation method of zirconium arsenate-bonded magnetic silicon spheres |
| WO2015166415A1 (en) * | 2014-04-28 | 2015-11-05 | Universidade De Aveiro | Chelator modified magnetic silica nanoparticles, their use and preparation |
| CN108070089A (en) * | 2016-11-14 | 2018-05-25 | 中国科学院大连化学物理研究所 | A kind of preparation method of the zirconium-based metallic organic framework material rich in zirconium ion |
| CN108070089B (en) * | 2016-11-14 | 2021-03-19 | 中国科学院大连化学物理研究所 | Preparation method of zirconium-based metal organic framework material rich in zirconium ions |
| CN116116385A (en) * | 2022-12-26 | 2023-05-16 | 北京青莲百奥生物科技有限公司 | Extraction of exosomes in blood and proteomic analysis method thereof |
| CN116116385B (en) * | 2022-12-26 | 2023-06-13 | 北京青莲百奥生物科技有限公司 | Extraction of exosomes in blood and proteomic analysis method thereof |
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