CN106807332B - A kind of hyaluronic acid decorated multi-stage nano particle and its preparation and application - Google Patents
A kind of hyaluronic acid decorated multi-stage nano particle and its preparation and application Download PDFInfo
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- CN106807332B CN106807332B CN201510862131.5A CN201510862131A CN106807332B CN 106807332 B CN106807332 B CN 106807332B CN 201510862131 A CN201510862131 A CN 201510862131A CN 106807332 B CN106807332 B CN 106807332B
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- 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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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- 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
- B01J20/28004—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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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
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- B01J2220/00—Aspects relating to sorbent materials
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- B01J2220/46—Materials comprising a mixture of inorganic and organic materials
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- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4806—Sorbents characterised by the starting material used for their preparation the starting material being of inorganic character
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- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character
- B01J2220/4825—Polysaccharides or cellulose materials, e.g. starch, chitin, sawdust, wood, straw, cotton
Abstract
The invention discloses a kind of hyaluronic acid decorated multi-stage nano particles, the multi-stage nano particle is the multi-layer silica dioxide particle of level-one or more, with rough surface, by hyaluronic acid decorated, the partial size of multi-stage nano particle is less than or equal to 1mm for the outer surface of multi-stage nano particle；Multi-stage nano particle is one of first grade silica particle, two layers of secondary silica grain, three layers of three-level silica dioxide granule and four layers fourth grade silica particle of single layer or a variety of.The invention also discloses the preparation method of the multi-stage nano particle and its applications in the enrichment and Mass Spectrometer Method of biological sample.The nano particle preparation cost is low, can in high volume make, synthesis step is simple；This grade of nanoscale rough, relative surface area is larger, and use is hyaluronic acid decorated, can remove the interference of other impurity proteins in biological sample (such as serum).
Description
Technical field
The present invention relates to Molecular Detection field more particularly to the enrichments and laser desorption ionogen of a kind of nano material
The Molecular Detection application technology of spectrum.
Background technique
It is traditional that albumen, gene and peptide fragment be directed primarily to the analysis method of serum, mass spectrometer system can it is highly sensitive,
High-resolution measures the charge-mass ratio of unknown molecular, and is surveyed by tandem mass spectrometry (tandem mass) to the molecule measured
Modification is translated thereafter in sequence, research.In principle, the molecular composition of Direct Identification biological sample is capable of in mass spectral analysis；
But in actual test, due to part albumen and the low abundance expression of peptide molecule and sample is excessively complicated, acquired in mass spectral analysis
Information it is often not comprehensive enough so that traditional detection method is very restricted, to limit it in medical diagnosis
Application.Therefore, in research method at this stage, the pretreatment of biological sample, which has become, determines mass spectral analysis efficiency
Final steps, wherein the sample pretreatment based on nano material is current hotspot field, and it is excellent to become the one kind tested and analyzed
Player's section.In actual biosystem, biological sample is usually sufficiently complex.The presence of various large biological molecules, these are all
Obstruction can be brought to the detection of protein molecular.
Summary of the invention
In view of the above drawbacks of the prior art, the multi-stage nano particle hyaluronic acid decorated the present invention provides one kind,
And be applied to and be enriched with low-abundance albumen from biological sample, assist the Mass Spectrometer Method of biological sample.
Hyaluronic acid (hyaluronic acid, HA) is by n-acetylglucosamine and β-D- glucuronic acid disaccharide unit
Threadiness acidic mucopolysaccharide made of alternately coupling, the major receptors of hyaluronic acid first is that CD44 molecule, in tumor locus or inflammation
The cell surface of disease tissue can over-express.The carrier for primarily now using HA to modify improves medicine as nano target carrier
The targeting of object transmitting.Present invention uses a kind of multi-stage nano materials, and rough surface, relative surface area is larger, using saturating
Bright matter acid specificity after surface modification, can replace the interactive protein molecular of antigen capture.By using hyaluronic acid
The granular materials auxiliary enrichment low-abundance protein of the multi-stage nano of modification, can overcome complexity in traditional serum molecules detection
Problem, quickly and accurately protein molecular in quantitative detection serum.
Technical scheme is as follows:
The present invention provides a kind of hyaluronic acid decorated multi-stage nano particle, which is level-one or more
Multi-layer silica dioxide particle has rough surface, and the outer surface of multi-stage nano particle is by hyaluronic acid decorated, multi-stage nano particle
Partial size be less than or equal to 1mm.
Further, multi-stage nano particle be the first grade silica particle of single layer, two layers of secondary silica grain,
One of three layers of three-level silica dioxide granule and four layers of fourth grade silica particle are a variety of.
The present invention also provides a kind of preparation methods of hyaluronic acid decorated multi-stage nano particle, comprising the following steps:
Step 1, the first grade silica particle that different-grain diameter is prepared using ethyl alcohol, ammonium hydroxide, TEOS, wherein first order dioxy
The particle size range of silicon carbide particle is 10-60nm, the particle size range of second level silica dioxide granule is 100-500nm, the third level two
The particle size range of silicon oxide particle is 0.85-3.5 μm；
It is step 2, first grade silica particle obtained in step 1 is ultrasonic in ethanol solution, until level-one titanium dioxide
Silicon particle is uniformly dispersed；
Step 3, by second level silica dioxide granule obtained in step 1 with 3- aminopropyl triethoxysilane in first
Amination modification, second level silica dioxide granule and the first grade silicon dioxide after then modifying amination are carried out in benzole soln
Particle is added in ultrapure water (i.e. resistivity reach 18M Ω * cm (25 degrees Celsius) water), and reacts under 20-100 degrees Celsius
12-24 hours, to be produced similar to the secondary silica grain of virus structure；
Step 4, by secondary silica obtained in third level silica dioxide granule obtained in step 1 and step 3
Grain carries out amination modification with 3- aminopropyl triethoxysilane respectively in toluene solution, and the after then modifying amination
Three-level silica dioxide granule and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and n-hydroxysuccinimide are added to richness
It in horse acid diformazan lipoprotein solution, is reacted 2-5 hours under 20-65 degrees Celsius, the secondary silica after adding amination modification
Particle carries out esterification under 20-65 degrees Celsius, to prepare three-level silica dioxide granule；
Step 5, by three-level silica dioxide granule obtained in step 4 and 3- aminopropyl triethoxysilane in toluene solution
Middle progress amination modification, three-level silica dioxide granule and succinic anhydride after then modifying amination are added to fumaric acid two
Carboxylated modification is carried out in formicester solution, three-level silica dioxide granule and 1- (3- dimethylamino after then modifying carboxylated
Propyl) -3- ethyl carbodiimide and n-hydroxysuccinimide be added in dimethyl fumarate solution, at 20-65 degrees Celsius
Lower reaction 2-5 hours adds the glass particle that particle size range is 0.5-1mm and carries out esterification, prepares fourth grade silica
Particle；
Step 6, by first grade silica particle, secondary silica grain obtained in step 1 to 5, three-level titanium dioxide
Silicon particle and fourth grade silica particle carry out amination modification with 3- aminopropyl triethoxysilane respectively in toluene solution,
Then hyaluronic acid and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and n-hydroxysuccinimide are added to rich horse
It in sour diformazan lipoprotein solution, is reacted 2-5 hours under 20-65 degrees Celsius, the first grade silica after adding amination modification
Grain, secondary silica grain, three-level silica dioxide granule and fourth grade silica particle carry out esterification preparation surface warp
Hyaluronic acid decorated multi-stage nano particle.
Further, it is used to prepare the preferred of the first grade silica particle of fourth grade silica particle are as follows: the first order two
The particle size range of silicon oxide particle is 10-20nm, and the particle size range of second level silica dioxide granule is 150-200nm, the third level
The particle size range of silica dioxide granule is 0.85-1.5 μm.
Further, the reaction temperature of amination modification is 80-120 degrees Celsius, and reflux time is 10-24 hours；
The reaction temperature of carboxylated modification is 20-60 degrees Celsius, and the reaction time is 12-24 hours；The reaction temperature of esterification is 20-
65 degrees Celsius.
The present invention also provides above-mentioned hyaluronic acid decorated multi-stage nano particle answering in Mass Spectrometer Method biological sample
With.
Further, hyaluronic acid decorated multi-stage nano particle is for being enriched with cancer cell proteins for Mass Spectrometer Method.
Further, cancer cell proteins are CD44 receptor protein.
The method that the present invention also provides a kind of to be enriched with CD44 receptor protein from serum, comprising the following steps:
Step 1 prepares hyaluronic acid decorated multi-stage nano particle according to the above method；
The hyaluronic acid decorated multi-stage nano particle prepared in step 1 is added in step 2, dilute serum sample (× 10),
It is sufficiently mixed (10-24 hours), the CD44 receptor protein after being enriched with.
The present invention also provides a kind of methods using the CD44 receptor protein in Mass Spectrometer Method serum, which is characterized in that
Method the following steps are included:
The preparation of step 1, instrument and reagent: laser desorption ionization mass spectrometry, using linear reflective mode, positive ion detection；
Step 2 is enriched with CD44 receptor protein according to the above method from serum；
Step 3 is centrifugally separating to obtain supernatant, carries out trypsin treatment；
Step 4: will treated sample point sample on mass spectrum target plate, dry at room temperature, using internal standard method to blood serum sample
In small molecule carry out quantitative analysis detection；
Step 5: mass spectrum testing result is analyzed, it was therefore concluded that.
The beneficial effects of the present invention are: the particle matrix preparation cost of multi-stage nano grade is low, can in high volume make, and closes
It is simple at step；This grade of nanoscale rough, relative surface area is larger, and use is hyaluronic acid decorated, can go
Except the interference of other impurity proteins in biological sample (such as serum).The present invention need to only consume a small amount of biological sample (such as blood
Clearly), inspection can quickly and accurately be quantified under the premise of the enrichment of hyaluronic acid decorated multi-stage nano material, lock out operation
Survey the specific cancer albumen in analysis biological sample (such as serum)；Entire detection process step is simple, at low cost, can be applied to
In clinic.
Below with reference to attached drawing, the invention will be further described, with absolutely prove the purpose of the present invention, technical characteristic and
Technical effect.
Detailed description of the invention
Fig. 1 is the SEM characterization picture for the multi-stage nano particle being prepared in preferred embodiment of the present invention: Fig. 1 a is 1mm
Nano particle SEM characterize picture；Three-level nano particle (surface has second level nano particle) SEM phenogram that Fig. 1 b is 10um
Piece；Fig. 1 c is that the 200nm of nano grain surface greater than 1000nm or so nanometer (second level nano particle) SEM characterizes picture；
Fig. 2 is the hyaluronic acid wave spectrum that FTIR spectrum figure marks the modification of multi-stage nano particle surface；
Fig. 3 is the unmodified multi-stage nano grain result with modification hyaluronic acid of Electrochemical Detection.
Specific embodiment
The present invention is described further with reference to the accompanying drawings and embodiments.
Step 1, the first grade silica particle that different-grain diameter is prepared using ethyl alcohol, ammonium hydroxide, TEOS, wherein first order dioxy
The particle size range of silicon carbide particle is 10-60nm, the particle size range of second level silica dioxide granule is 100-500nm, the third level two
The particle size range of silicon oxide particle is 0.85-3.5 μm；
It is step 2, first grade silica particle obtained in step 1 is ultrasonic in ethanol solution, until level-one titanium dioxide
Silicon particle is uniformly dispersed；
Step 3, by second level silica dioxide granule obtained in step 1 with 3- aminopropyl triethoxysilane in first
Amination modification, second level silica dioxide granule and the first grade silicon dioxide after then modifying amination are carried out in benzole soln
Particle is added in ultrapure water, and reacts 12-24 hours under 20-100 degrees Celsius, to prepare secondary silica grain；
Step 4, by secondary silica obtained in third level silica dioxide granule obtained in step 1 and step 3
Grain carries out amination modification with 3- aminopropyl triethoxysilane respectively in toluene solution, and the after then modifying amination
Three-level silica dioxide granule and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and n-hydroxysuccinimide are added to richness
It in horse acid diformazan lipoprotein solution, is reacted 2-5 hours under 20-65 degrees Celsius, the secondary silica after adding amination modification
Particle carries out esterification under 20-65 degrees Celsius, to prepare three-level silica dioxide granule；
Step 5, by three-level silica dioxide granule obtained in step 4 and 3- aminopropyl triethoxysilane in toluene solution
Middle progress amination modification, three-level silica dioxide granule and succinic anhydride after then modifying amination are added to fumaric acid two
Carboxylated modification is carried out in formicester solution, three-level silica dioxide granule and 1- (3- dimethylamino after then modifying carboxylated
Propyl) -3- ethyl carbodiimide and n-hydroxysuccinimide be added in dimethyl fumarate solution, at 20-65 degrees Celsius
Lower reaction 2-5 hours adds the glass particle that particle size range is 0.5-1mm and carries out esterification, prepares fourth grade silica
Particle；
Step 6, by first grade silica particle, secondary silica grain obtained in step 1 to 5, three-level titanium dioxide
Silicon particle and fourth grade silica particle carry out amination modification with 3- aminopropyl triethoxysilane respectively in toluene solution,
Then hyaluronic acid and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and n-hydroxysuccinimide are added to rich horse
It in sour diformazan lipoprotein solution, is reacted 2-5 hours under 20-65 degrees Celsius, the first grade silica after adding amination modification
Grain, secondary silica grain, three-level silica dioxide granule and fourth grade silica particle carry out esterification preparation surface warp
Hyaluronic acid decorated multi-stage nano particle.
Synthesize from small particle to big partial size various two layers of second level titanium dioxide step by step using various first grade silica particles
Silicon particle, three layers of three-level silica dioxide granule and four layers of fourth grade silica particle.According to stability and relative surface area
Increased optimization criteria is used to prepare the preferred of the first grade silica particle of fourth grade silica particle are as follows: the first order two
The particle size range of silicon oxide particle is 10-20nm, and the particle size range of second level silica dioxide granule is 150-200nm, the third level
The particle size range of silica dioxide granule is 0.85-1.5 μm.It is obtained using the first grade silica particle preparation in these particle size ranges
The fourth grade silica particle arrived with optimal stability and is suitable for capturing the surface area of specific protein.
In above-mentioned preparation step, the reaction temperature of amination modification is 80-120 degrees Celsius, reflux time 10-24
Hour；The reaction temperature of carboxylated modification is 20-60 degrees Celsius, and the reaction time is 12-24 hours；The reaction temperature of esterification
It is 20-65 degrees Celsius.
The characterization of multi-stage nano material:
Characterization instrument has: obtaining transmission electron microscope picture, high-resolution-ration transmission electric-lens using JEOL JEM-2100F instrument
Picture and selective electron diffraction style；Scanning electron microscope example is prepared using silicon wafer, and is obtained by Hitachi S-4800 instrument
Obtain scanning electron microscopic picture；Contact angle uses EasyDrop device KRUSS GmbH, Germany to obtain.
Characterization result are as follows:
Nano-particle material obtained is spherical shape, and final nanoparticle size is less than or equal to 1mm.Pass through transmission electron microscope picture
Piece can be seen that granular materials with subunit structure, and by choosing the amplification of granular materials fringe region as can be seen that table
Face more nano-particle materials by being made of.According to the scanning electron microscopic picture (as shown in Figure 1) measured, it can be seen that particle
Coarse shape, and size uniformity is presented in material surface.By FTIR spectrum it is found that the nano-particle material of functionalized modification
There is good absorption peak in 1480 regions, indicate that hyaluronic acid has been modified in multi-stage nano material surface.Fig. 2 is that Fourier is red
External spectrum figure marks the hyaluronic acid wave spectrum of multi-stage nano particle surface modification.Fig. 3 is that Electrochemical Detection is unmodified
(Control) and modification various concentration (1ng/ml-104Ng/ml) after the multi-stage nano particle capture CD44 albumen of hyaluronic acid
The variation of electric current, it can be found that the multi-stage nano material current significant change after modification, illustrates through hyaluronic acid decorated multistage
Nano particle has been successfully acquired CD44 albumen.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Claims (8)
1. a kind of preparation method of hyaluronic acid decorated multi-stage nano particle, which is characterized in that the preparation method include with
Lower step:
Step 1, the first grade silica particle that different-grain diameter is prepared using ethyl alcohol, ammonium hydroxide, TEOS, wherein the first grade silicon dioxide
The particle size range of particle is 10-60nm, the particle size range of second level silica dioxide granule is 100-500nm, third level titanium dioxide
The particle size range of silicon particle is 0.85-3.5 μm；
It is step 2, first grade silica particle obtained in step 1 is ultrasonic in ethanol solution, until the level-one two
Silicon oxide particle is uniformly dispersed；
It is step 3, second level silica dioxide granule obtained in step 1 is molten in toluene with 3- aminopropyl triethoxysilane
Amination modification is carried out in liquid, the second level silica dioxide granule and the first order dioxy after then modifying amination
Silicon carbide particle is added in ultrapure water, and reacts 12-24 hours under 20-100 degrees Celsius, to prepare secondary silica grain；
Step 4, by the second level titanium dioxide obtained in third level silica dioxide granule obtained in step 1 and step 3
Silicon particle carries out amination modification with 3- aminopropyl triethoxysilane respectively in toluene solution, after then modifying amination
The third level silica dioxide granule and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and n-hydroxysuccinimide
It is added in dimethyl fumarate solution, is reacted 2-5 hours under 20-65 degrees Celsius, it is described after adding amination modification
Secondary silica grain carries out esterification under 20-65 degrees Celsius, to prepare three-level silica dioxide granule；
Step 5, by three-level silica dioxide granule obtained in step 4 and 3- aminopropyl triethoxysilane in toluene solution
Middle progress amination modification, the three-level silica dioxide granule and succinic anhydride after then modifying amination are added to rich horse
Carboxylated modification is carried out in sour diformazan lipoprotein solution, the three-level silica dioxide granule and 1- (3- after then modifying carboxylated
Dimethylamino-propyl) -3- ethyl carbodiimide and n-hydroxysuccinimide be added in dimethyl fumarate solution, in 20-
It is reacted 2-5 hours under 65 degrees Celsius, adds the glass particle that particle size range is 0.5-1mm and carry out esterification, prepare level Four
Silica dioxide granule；
Step 6, by first grade silica particle, the secondary silica grain, described three obtained in step 1 to 5
Grade silicon dioxide particle and the fourth grade silica particle respectively with 3- aminopropyl triethoxysilane in toluene solution into
Row amination modification, then by hyaluronic acid and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide and N- hydroxysuccinimidyl acyl
Imines is added in dimethyl fumarate solution, is reacted 2-5 hours under 20-65 degrees Celsius, after adding amination modification
The first grade silica particle, the secondary silica grain, the three-level silica dioxide granule and the level Four dioxy
Silicon carbide particle carries out esterification preparation surface through the hyaluronic acid decorated multi-stage nano particle.
2. preparation method according to claim 1, which is characterized in that be used to prepare the institute of the fourth grade silica particle
State the preferred of first grade silica particle are as follows: the particle size range of first order silica dioxide granule is 10-20nm, second level titanium dioxide
The particle size range of silicon particle is 150-200nm, and the particle size range of third level silica dioxide granule is 0.85-1.5 μm.
3. preparation method according to claim 1, which is characterized in that the reaction temperature of the amination modification is 80-120
Degree Celsius, reflux time is 10-24 hours；The reaction temperature of the carboxylated modification is 20-60 degrees Celsius, the reaction time
It is 12-24 hours；The reaction temperature of the esterification is 20-65 degrees Celsius.
4. the multi-stage nano particle of preparation method preparation described in any one of claim 1 to 3 is in Mass Spectrometer Method biology
Application in sample, which is characterized in that the multi-stage nano particle is the multi-layer silica dioxide particle of level-one or more, the multistage
Nano particle has rough surface, and the outer surface of the multi-stage nano particle is by hyaluronic acid decorated, the multi-stage nano particle
Partial size be less than or equal to 1mm.
5. application according to claim 4, which is characterized in that the hyaluronic acid decorated multi-stage nano particle is for richness
Collect cancer cell proteins to be used for Mass Spectrometer Method.
6. application according to claim 5, which is characterized in that the cancer cell proteins are CD44 receptor protein.
7. a kind of method for being enriched with CD44 receptor protein from serum, which is characterized in that the described method comprises the following steps:
Step 1 prepares hyaluronic acid decorated multi-stage nano particle according to the method for claim 1；
The hyaluronic acid decorated multi-stage nano particle prepared in step 1 is added in step 2, dilute serum sample, sufficiently mixed
It closes, the CD44 receptor protein after being enriched with.
8. a kind of method using the CD44 receptor protein in Mass Spectrometer Method serum, which is characterized in that the method includes following
Step:
The preparation of step 1, instrument and reagent: laser desorption ionization mass spectrometry, using linear reflective mode, positive ion detection；
Step 2 is enriched with the CD44 receptor protein from serum according to the method for claim 7；
Step 3 is centrifugally separating to obtain supernatant, carries out trypsin treatment；
Step 4: will treated sample point sample on mass spectrum target plate, dry at room temperature, using internal standard method in blood serum sample
Small molecule carries out quantitative analysis detection；
Step 5: mass spectrum testing result is analyzed, it was therefore concluded that.
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| CN201510862131.5A CN106807332B (en) | 2015-11-30 | 2015-11-30 | A kind of hyaluronic acid decorated multi-stage nano particle and its preparation and application |
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| CN201510862131.5A CN106807332B (en) | 2015-11-30 | 2015-11-30 | A kind of hyaluronic acid decorated multi-stage nano particle and its preparation and application |
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| CN106807332B true CN106807332B (en) | 2019-05-14 |
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| CN103157441A (en) * | 2013-03-29 | 2013-06-19 | 中国科学院合肥物质科学研究院 | Fluorine removal agent with fluorine ion absorption/desorption function through pH value regulation and control and preparation method thereof |
| CN104176740A (en) * | 2010-05-21 | 2014-12-03 | 格雷斯股份有限两合公司 | Porous inorganic oxide particles and methods of making and using the same |
| CN104874359A (en) * | 2015-05-28 | 2015-09-02 | 吉林大学 | Preparing method of calixarene modified magnetic material |
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| CN104176740A (en) * | 2010-05-21 | 2014-12-03 | 格雷斯股份有限两合公司 | Porous inorganic oxide particles and methods of making and using the same |
| CN103157441A (en) * | 2013-03-29 | 2013-06-19 | 中国科学院合肥物质科学研究院 | Fluorine removal agent with fluorine ion absorption/desorption function through pH value regulation and control and preparation method thereof |
| CN104874359A (en) * | 2015-05-28 | 2015-09-02 | 吉林大学 | Preparing method of calixarene modified magnetic material |
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| 温敏性异丙基丙烯酰胺-二氧化硅透明质酸核壳型杂化微凝胶的合成和体积相变行为;左雪芹;《功能高分子学报》;20100930;第23卷(第3期);第266页，第4段，第267页，图7，第3段 |
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