CN108287234A - Nano immune magnetic bead and its preparation method and application - Google Patents
Nano immune magnetic bead and its preparation method and application Download PDFInfo
- Publication number
- CN108287234A CN108287234A CN201710173694.2A CN201710173694A CN108287234A CN 108287234 A CN108287234 A CN 108287234A CN 201710173694 A CN201710173694 A CN 201710173694A CN 108287234 A CN108287234 A CN 108287234A
- Authority
- CN
- China
- Prior art keywords
- magnetic bead
- nano immune
- immune magnetic
- preparation
- glucan
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention discloses a kind of nano immune magnetic beads and its preparation method and application.The preparation method of the nano immune magnetic bead of the present invention can prepare the glucan coating Fe of 50nm ranks3O4And/or γ Fe2O3Nano immune magnetic bead, the grain size of the obtained nano immune magnetic bead is small, and particle diameter distribution is uniform, and stability is good, can be to avoid magnetic bead aggregation and sedimentation.In addition, by synthesizing obtained nano immune magnetic bead coupled antibody, it is ensured that the amount of every batch of magnetic bead surfaces antibody is almost the same, favorable repeatability, it sorts out positive cell purity and reaches 90% or more, impurity content is low, can be widely used in the specificity sorting and separation process of cell.The preparation method of the nano immune magnetic bead is quick, easy, and cost is relatively low, can wide popularization and application.
Description
Technical field
The present invention relates to cell engineering fields, more particularly, to a kind of nano immune magnetic bead and its preparation method and application.
Background technology
Cell sorting has very important effect in clinical diagnosis, cellular immunotherapy etc..In terms of clinical diagnosis,
Such as：Fetal cell is detached to judge whether fetus has genetic defect；Cancer cell is detached to realize the early diagnosis of cancer and control
It treats；Hematopoietic stem cell transplantation can be used as the major measure of hematopoiesis support treatment after malignant tumour Large Dose Irradiation, chemotherapy.Cell
In terms of immunization therapy, immunocyte such as is isolated from peripheral blood mononuclear cells, in vitro using cell factor by its
Activation becomes cytokine-induced killer cell cell, as DC-CIK cells, or use technique for gene engineering give T cell to be added
One can tumor cell and at the same time the chimeric antibody of activation T cell makes its defeated ex vivo again after large amplification in vitro
It is interior, to kill the tumour cell with corresponding specific antigen, such as CAR-T cells.
Cell sorting method mainly has two major classes：One kind is based on being established there are the difference of sedimentation coefficient between cell mass
The density-gradient centrifugation method (Density gradient centrifugation) to get up；Another kind of is special based on Immune discrimination
The method of property, including fluorescence-activated cell sorting method (Fluorescence-activated cell sorting, FACS) and
Magnetic activated cell separating method (is also known as magnetic bead sorting method, Magnetic-activated cell separation, MACS).
Density-gradient centrifugation method is simple and practicable, but isolated cell purity is low, and the mark of cell surface is indefinite, specificity compared with
Difference, therefore, oneself is less at present uses such method.The cell purity of fluidic cell sorting gained is high, the rate of recovery is high, it is dirty to be not easy
Dye, but the equipment needed for the method is costly, time-consuming, larger to cytositimulation, and need high-caliber technical support and profession
Operating personnel.In addition, this method can only sort a cell sample whithin a period of time.
Magnetic bead sorting method is a kind of excellent cell sorting techniques to grow up the 1970s.It is related to immune
Magnetic bead is a kind of magnetic-particle being coated with monoclonal antibody, can be with the antigen binding of target cell surface.In externally-applied magnetic field, lead to
It crosses the target cell that antibody is connected with magnetic bead to be adsorbed and be trapped in sorting column, the cell without this kind of surface antigen is due to cannot be with
Magnetic bead is connected without magnetism, cannot be stopped in magnetic field, to achieve the purpose that cell enrichment purifies.The use being commercialized at present
Mainly include the superparamagnetism magnetic of German U.S. day Ni (Miltenyi) nanoscale glucan cladding in the magnetic bead product of cell sorting
Pearl and the U.S. wear promise (Dynal) micron order polystyrene magnetic beads.And for wearing promise micron order polystyrene magnetic beads, U.S. day
The magnetic bead of the 50nm of Ni or so has the advantage that：1, after magnetic bead and cell combination, it is not necessary that magnetic bead is disintegrated down, and to cell
Physiology and activity have no effect；2, glucan package nanometer magnetic bead is degradable, good biocompatibility, to cell without any poison
Property；3, flow cytometer showed can be carried out without being affected substantially, on subsequent experimental without influence by being attached with the target cell of little magnetic bead；4, it sorts
Target cell purity out is high, can avoid the interference of other non-specific cellulars.
Although the 50nm magnetic bead better performances of U.S. day Ni, expensive, domestic research institution and enterprise substantially according to
By external import, this also allows medium-sized and small enterprises to hang back.Although the domestic magnetic also developed successively for cell analysis, protein purification
Pearl, but these magnetic bead grain sizes are mainly distributed on micron order, and also wrapping layer is concentrated mainly on organic and inorganic substances, and it is acquired
The biocompatibility and degradability of magnetic bead are poor, are difficult to apply in organism.And some coated glucides are to adopt
It is wrapped up with two-step method, is to use Co deposited synthesis nanometer Magnetic particles, then wrapped up with glucan on surface first, thus
The nano-particle that can make is easy aggregation, and dispersibility is poor, and such as application No. is the patent applications of CN200710176211
In the method mentioned；And the nano-particle of one-step method glucan package is used to be repaiied in group though obtained stability is preferable
Cyanogen bromide is used during decorations, however cyanogen bromide has severe toxicity to also limit it using (such as Clonis YD, Small DAP.High-
performance dye-ligand Chromatograph.In Reactive in Protein and Enzyme
Technology(M).London:Macmillan Press, 1987.87-100).
Invention content
Based on this, it is necessary to provide that a kind of grain size is smaller, stability is good and the nano immune magnetic bead of preparation method simplicity and
Preparation method and application.
The technical solution that the present invention solves above-mentioned technical problem is as follows.
A kind of preparation method of nano immune magnetic bead, which is characterized in that include the following steps：
Step 1：Anaerobic reaction solution is prepared, being added in the anaerobic reaction solution has glucan, Fe2+And Fe3+,
In, Fe2+And Fe3+The sum of concentration be 0.03mol/l~1mol/l, Fe3+With Fe2+Molar ratio be 0.25:1~4:1, Fe2+
And Fe3+The sum of the amount of substance and the ratio of the amount of the substance of glucan be 90:1~15:1, the glucan is poly- for hydroxyl Portugal
Sugar or carboxyl glucan；
Step 2：The anaerobic reaction solution is stirred to react 10min with 500~1200rpm/min at 50 DEG C, then is added
It is 9.0~11 to enter ammonium hydroxide to adjust acid-base value to pH, continues to be stirred to react 30min~2h at 50~100 DEG C；
Step 3：It is cooled to room temperature after reaction, larger particle, the solution of collection are adjusted with acid in centrifugation removal solution
Acid-base value is saved to neutrality, then uses 0.22 μm of hydrophilic filter membrane filtering further to clean, obtained filtrate is after purification to obtain the final product
Glucan is coated with Fe3O4And/or γ Fe2O3The nano immune magnetic bead.
When step 1 configures solution, can first be selected into solvent in anaerobic item by nitrogen deoxygenation, subsequent preparation process
Under part, as reacted in nitrogen or atmosphere of inert gases.
The molecular weight of the glucan is 20kDa~100kDa in one of the embodiments,.
Further, the molecular weight of the glucan is 20kDa, 40kDa or 100kDa in one of the embodiments,.
Fe in one of the embodiments,2+And Fe3+The sum of concentration be 0.09mol/l.
Fe in one of the embodiments,3+With Fe2+Molar ratio be 2:1.
Fe in one of the embodiments,2+And Fe3+The sum of the amount of substance and the ratio of the amount of the substance of glucan be
90:1、45:1、30:1、18:1 or 15:1.Further, the sum of the amount of substance of Fe2+ and Fe3+ in one of the embodiments,
Ratio with the amount of the substance of glucan is 45:1.
In one of the embodiments, in the step 2, be by the anaerobic reaction solution at 50 DEG C with
1000rpm/min is stirred to react 10min, and it is 10 to add ammonium hydroxide and adjust acid-base value to pH, continues to be stirred to react 1h at 70 DEG C.
The concentrated ammonia liquor of a concentration of 5mol/L~15mol/L can be selected in ammonium hydroxide, and the addition of concentrated ammonia liquor can react molten according to every 100ml anaerobics
8ml~50ml is added in liquid, can such as select that 8ml, 12ml, 16ml, 24ml or 50ml is added, dense in one of the embodiments,
The amount that 12ml can be added in the addition of ammonium hydroxide according to every 100ml anaerobics reaction solution is added.
In one of the embodiments, in the step 3, the rotating speed of the centrifugation is 3000rpm, and the time is 5min.
In one of the embodiments, in the step 3, using a concentration of 0.1M~2.0M HCl or glacial acetic acid into
Row acid-base value adjust, such as can be used a concentration of 1.0M HCl adjust acid-base value to pH be 7.0.
In one of the embodiments, in the step 3, the purifying was (Germany U.S. day Ni, MS points of sorting column
Select column) purifying, to remove wherein free glucan.
A kind of nano immune magnetic bead is prepared into using the preparation method of the nano immune magnetic bead described in any of the above-described embodiment
It arrives.
The nano immune magnetic bead being prepared can tune to a concentration of 6mg/ml be stored in it is spare at 4 DEG C.
Application of the above-mentioned nano immune magnetic bead in cell sorting.
As the application includes that nano immune magnetic bead is combined and used with specific antibody in one of the embodiments,
The nano immune magnetic bead of specific antibody is combined with target cell by specific antibody-target cell surface antigen binding, and
The step of being screened in magnetic field.For carboxyl glucan, directly it can be combined with specific antibody；For hydroxyl glucan,
Aldehydedodextrans can be combined with atopic antibody first by hydroxyl dextran oxidation at aldehydedodextrans.
In one of the embodiments, for the coated nano immune magnetic bead of hydroxyl glucan, can by the following method by
Hydroxyl dextran oxidation is aldehydedodextrans：
Sodium metaperiodate is added into the coated nano immune magnetic bead solution of hydroxyl glucan, wherein the addition of sodium metaperiodate
Amount meets：Nano immune magnetic bead:Sodium metaperiodate=0.125~8:1 (mass ratio), room temperature, which is protected from light, is stirred to react 30min~3h, instead
Column separating purification is crossed after answering and removes the glucan that dissociates, and is that 7.0~9.0 dobell's solutions wash with 10mM~100mM, pH
Afterwards to get.Further, a concentration of 4mg/ml of the also coated nano immune magnetic bead of adjustable aldehydedodextrans is saved backup.
Further, the mass ratio of nano immune magnetic bead and sodium metaperiodate can be 8 in one of the embodiments,:1、4:
1、2:1、1:1、1:2、1:3、1:4 or 1:8, wherein 1:1 is preferred mass ratio.
Further, it is protected from light in one of the embodiments, and is stirred to react the time as 1h.
Further, it is to be washed 3 times for 8.5 dobell's solutions using 20mM, pH in one of the embodiments,.
Aldehydedodextrans or the coated nano immune magnetic bead of carboxyl glucan are combined with specific antibody can according to but not
Be limited to following steps progress, such as in one of the embodiments, for the aldehydedodextrans of concentration 4mg/ml that save backup or
The coated nano immune magnetic bead of carboxyl glucan can use 100 μ l, be after 8.5 Boratexes dilute one times, according to receiving with 20mM, pH
Rice immunomagnetic beads:Antibody mass ratio is 1~100:Antibody is added in 1 ratio, and normal-temperature reaction vibrates mixing 1h~12h, and reaction terminates
Afterwards, excessive NaBH is added into reaction system430min~3h is reacted, column separating purification is crossed, is washed repeatedly with PBS buffer solution,
Phosphate buffer is added to save backup in 4 DEG C.Other volumes can be proportionally adjusted.
It further, in one of the embodiments, can be according to nano immune magnetic bead:Antibody mass ratio is 5:1、15:1、30:
1 or 100:The antibody of concentration 5mg/ml is added in 1 ratio.Wherein, nano immune magnetic bead:Antibody mass is than preferably 15:1, often
The time of temperature reaction oscillation mixing is preferably 6h.
It further, in one of the embodiments, can be according to nano immune magnetic bead:NaBH4Mass ratio is 20:1、10:
1、1:10 or 1:The NaBH of a concentration of 0.25M is added in 20 ratios4.Wherein, preferred ratio is 10:1, the preferred reaction time is
1h。
The glucan that 50nm ranks can be prepared by the preparation method of above-mentioned nano immune magnetic bead is coated with Fe3O4And/or γ
Fe2O3Nano immune magnetic bead, the grain size of the obtained nano immune magnetic bead is small, and particle diameter distribution is uniform, and stability is good, can
To avoid magnetic bead aggregation and sedimentation.In addition, by synthesizing obtained nano immune magnetic bead coupled antibody, it is ensured that every batch of magnetic bead
The amount of surface antibody is almost the same, favorable repeatability, sorts out positive cell purity and reaches 90% or more, and impurity content is low,
It can be widely used in the specificity sorting and separation process of cell.The preparation method of the nano immune magnetic bead is quick, easy, at
This is relatively low, can wide popularization and application.
Description of the drawings
Fig. 1 is the particle diameter distribution testing result figure for the nano immune magnetic bead that embodiment 1 is prepared；
Fig. 2 is the SEM figures of hydroxyl nano immune magnetic bead made from embodiment 4；
Fig. 3 is the fluorescent staining result figure for the cd4 t cell that embodiment 5 sorts out.
Specific implementation mode
To facilitate the understanding of the present invention, below with reference to relevant drawings to invention is more fully described.In attached drawing
Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes
The embodiment of description.Keep the understanding to the disclosure more thorough on the contrary, purpose of providing these embodiments is
Comprehensively.
Unless otherwise defined, all of technologies and scientific terms used here by the article and belong to the technical field of the present invention
The normally understood meaning of technical staff is identical.Used term is intended merely to description tool in the description of the invention herein
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the Listed Items of pass.
The synthesis of the 1 coated nano immune magnetic bead of 50nm hydroxyl glucans of embodiment
8.0g hydroxyl glucans (molecular weight 40kDa) are weighed first, are dissolved into 100ml deionized waters, letting nitrogen in and deoxidizing
After 10min, 1.62g FeCl are added3.6H2O and 0.62g FeCl2.4H2O, 1000rpm/min stir 50 DEG C of heating water baths
After 10min, be added 12ml a concentration of 15mol/L concentrated ammonia liquor adjust acid-base value to pH be 10.0；Temperature rise to 70 DEG C heat preservation after
After continuous reaction 1h, stops reaction and be cooled to room temperature；3000rpm centrifuges 5min by after bulky grain is centrifuged off in solution, uses 1.0M
HCl adjusts acid-base value to neutrality, then is filtered with 0.22 μm of hydrophilic filter membrane, and obtained filtrate is sorted with Germany U.S. day Ni MS
Column removes the glucan to dissociate in solution；6mg/mL hydroxyl glucan nanometer magnetic beads are taken to be diluted to 1mg/ml with ultra-pure water, DLS pairs
Its grain size is characterized, and is averaged three times, you can obtains the coated nanometer magnetic bead of 50nm hydroxyl glucans, 4 DEG C of preservations.
As shown in Figure 1, the average grain diameter of the obtained coated nanometer magnetic bead of hydroxyl glucan is 59.50nm, and hydroxyl magnetic
Spherical individual particle distribution, size uniform is presented in pearl.
The stability result for the coated nanometer magnetic bead of hydroxyl glucan that the following table 1 is.
1 hydroxyl magnetic bead different time droplet measurement of table
| Average grain diameter | Peak diameter | PDI | |
| Place January | 59.50 | 72.2 | 0.172 |
| Place March | 58.65 | 71.5 | 0.174 |
| Place June | 60.12 | 72.1 | 0.171 |
As can be seen from Table 1 hydroxyl magnetic bead can be stored at 4 DEG C will not occur aggregation between the half a year above nano-particle and
Nano-particle performance is influenced, providing stability for subsequent cell sort applications ensures.
2 glucan of embodiment：Iron ion/ferrous ion is 1:The 90 synthesis coated nano immune magnetics of 50nm hydroxyl glucans
Pearl
4.4g hydroxyl glucans (molecular weight 40kDa) are weighed first, are dissolved into 100ml deionized waters, letting nitrogen in and deoxidizing
After 10min, 1.62g FeCl are added3.6H2O and 0.62g FeCl2.4H2O, 1000rpm/min stir 50 DEG C of heating water baths
After 10min, be added 12ml a concentration of 15mol/L concentrated ammonia liquor adjust acid-base value to pH be 10.0；Temperature rise to 70 DEG C heat preservation after
After continuous reaction 1h, stops reaction and be cooled to room temperature；3000rpm centrifuges 5min by after bulky grain is centrifuged off in solution, uses 1.0M
HCl adjusts acid-base value to neutrality, then is filtered with 0.22 μm of hydrophilic filter membrane, and obtained filtrate is sorted with Germany U.S. day Ni MS
Column removes the glucan to dissociate in solution；6mg/mL hydroxyl glucan nanometer magnetic beads are taken to be diluted to 1mg/ml with ultra-pure water, DLS pairs
Its grain size is characterized, and is averaged three times, you can obtains the coated nanometer magnetic bead of 58nm hydroxyl glucans, 4 DEG C of preservations.
3 glucan of embodiment：Iron ion/ferrous ion is 1:The 15 synthesis coated nano immune magnetics of 50nm hydroxyl glucans
Pearl
24g hydroxyl glucans (molecular weight 40kDa) are weighed first, are dissolved into 100ml deionized waters, letting nitrogen in and deoxidizing
After 10min, 1.62g FeCl are added3.6H2O and 0.62g FeCl2.4H2O, 1000rpm/min stir 50 DEG C of heating water baths
After 10min, be added 12ml a concentration of 15mol/L concentrated ammonia liquor adjust acid-base value to pH be 10.0；Temperature rise to 70 DEG C heat preservation after
After continuous reaction 1h, stops reaction and be cooled to room temperature；3000rpm centrifuges 5min by after bulky grain is centrifuged off in solution, uses 1.0M
HCl adjusts acid-base value to neutrality, then is filtered with 0.22 μm of hydrophilic filter membrane, and obtained filtrate is sorted with Germany U.S. day Ni MS
Column removes the glucan to dissociate in solution；6mg/mL hydroxyl glucan nanometer magnetic beads are taken to be diluted to 1mg/ml with ultra-pure water, DLS pairs
Its grain size is characterized, and is averaged three times, you can obtains the coated nanometer magnetic bead of 55nm hydroxyl glucans, 4 DEG C of preservations.
The SEM of the 4 coated nanometer magnetic bead of 50nm hydroxyl glucans of embodiment schemes
Take 0.1mg/mL hydroxyl nano immune magnetic bead (embodiment 1, the glucan that 10 μ L were diluted with water：Iron ion/ferrous iron
Ion is 1:45) it drips on copper mesh, waits for that it after natural drying, is characterized with SEM, the results are shown in Figure 2：It can from left figure
It is distributed to hydroxyl magnetic bead in single-size shape, right figure sees that most of magnetic bead grain size is 60nm or so, basic with DLS testing results
Unanimously.
5 nano immune magnetic bead coupled antibody of embodiment
Take made from 500 μ l embodiments 1 that the coated nano immune magnetic bead of 6mg/ml hydroxyl glucans is in EP pipes, according to height
Sodium iodate:Nano immune magnetic bead=1:Sodium periodate solution is added in 1 mass ratio, after room temperature is protected from light oscillating reactions 1h, using magnetic
The impurity that column method removes nano-particle institute band was detached, is finally eluted, is adjusted with the 20mM pH dobell's solutions for being 8.5
A concentration of 5mg/mL of nano immune magnetic bead.
According to nano immune magnetic bead:Specific antibody=15:Specific antibody is added to magnetic bead solution by 1 mass ratio
In, oscillation mixing reacts 6h, excessive sodium borohydride is then added, such as according to specific antibody:Sodium borohydride=1:1 quality
Than being added, the nano immune magnetic bead of specific antibody coupling is obtained with sodium borohydride reduction, again used column method purification specificity
Antibody-coupled magnetic beads, 4 DEG C of preservations.
6 CD4 antibody-coupled magnetic beads of embodiment are extracted for people's cd4 t cell
1. people's new blood that anti-coagulants is added in 20ml dilutes 2 times with PBS (phosphate buffer), according to blood:Lymph
Separating liquid=1:1 volume ratio tiles the blood diluted onto lymph separating liquid；Peripheral blood list is obtained using centrifugal method
A nucleus, adjustment cell concentration are 3 × 107A cell/ml；
2. starching 100 μ l 3 × 107The cell suspension of a cell/ml and the nano immune magnetic bead of 5 μ l CD4 antibody couplings (make
Method is prepared used in embodiment 2, uses CD4 antibody as specific antibody) it is stood in 4 DEG C with after liquid-transfering gun mixing
15min；
3. supernatant is abandoned in 1200rpm, 5min centrifugation, the resuspension of 500 μ l buffer solutions is added into cell precipitation；
4. cleaning cell 3 times with U.S. day Ni sorting columns, every time after the washing of 500 μ l buffer solutions, 1ml buffer solutions are added by cell
It rinses in collection to EP pipes；
5. cell is centrifuged with 1200rpm, 5min and abandons supernatant, the resuspension of 100 μ l buffer solutions is added into cell precipitation, is added 10
4 DEG C of incubation 5min of CD4 antibody of μ l PE labels；
6. supernatant is abandoned in 1200rpm, 5min centrifugation, the resuspension of 100 μ lPBS buffer solutions, fluorescence microscopy are added into cell precipitation
Sem observation cell fluorescence.
The results are shown in Figure 3, after the cell come out as seen from Figure 3 with immunological magnetic bead sorting carries out fluorescent marker, shows
See that the cell with fluorescence reaches 90% under micro mirror.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of preparation method of nano immune magnetic bead, which is characterized in that include the following steps：
Step 1：Anaerobic reaction solution is prepared, being added in the anaerobic reaction solution has glucan, Fe2+And Fe3+, wherein Fe2+
And Fe3+The sum of concentration be 0.03mol/l~1mol/l, Fe3+With Fe2+Molar ratio be 0.25:1~4:1, Fe2+And Fe3+'s
The ratio of the sum of amount of substance and the amount of the substance of glucan is 90:1~15:1, the glucan is hydroxyl glucan or carboxyl
Glucan；
Step 2：The anaerobic reaction solution is stirred to react 10min with 500~1200rpm/min at 50 DEG C, adds ammonia
It is 9.0~11 that water, which adjusts acid-base value to pH, continues to be stirred to react 30min~2h at 50~100 DEG C；
Step 3：It is cooled to room temperature after reaction, larger particle, the solution of collection are adjusted with acid acid in centrifugation removal solution
Then it is poly- up to Portugal after purification to filter the filtrate obtained further to clean to neutrality with 0.22 μm of hydrophilic filter membrane for basicity
Glycolyx Fe3O4And/or γ Fe2O3The nano immune magnetic bead.
2. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that the molecular weight of the glucan is
20kDa~100kDa.
3. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that the molecular weight of the glucan is
20kDa, 40kDa or 100kDa.
4. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that Fe2+And Fe3+The sum of concentration be
0.09mol/l。
5. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that Fe3+With Fe2+Molar ratio be 2:
1。
6. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that Fe2+And Fe3+Substance amount it
Ratio with the amount of the substance with glucan is 90:1、45:1、30:1、18:1 or 15:1.
7. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that in the step 2, be by
The anaerobic reaction solution at 50 DEG C with 1000rpm/min be stirred to react 10min be and then added ammonium hydroxide adjust acid-base value to
PH is 10, continues to be stirred to react 1h at 70 DEG C.
8. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that described in the step 3
The rotating speed of centrifugation is 3000rpm, and the time is 5min.
9. a kind of nano immune magnetic bead, which is characterized in that using such as nano immune magnetic according to any one of claims 1 to 8
The preparation method of pearl is prepared.
10. application of the nano immune magnetic bead as claimed in claim 9 in cell sorting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710173694.2A CN108287234A (en) | 2017-03-22 | 2017-03-22 | Nano immune magnetic bead and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710173694.2A CN108287234A (en) | 2017-03-22 | 2017-03-22 | Nano immune magnetic bead and its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108287234A true CN108287234A (en) | 2018-07-17 |
Family
ID=62831361
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710173694.2A Pending CN108287234A (en) | 2017-03-22 | 2017-03-22 | Nano immune magnetic bead and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108287234A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444401A (en) * | 2018-12-12 | 2019-03-08 | 郑州安图生物工程股份有限公司 | A kind of preparation method of magnetic microparticle chemiluminescence product |
| CN114470183A (en) * | 2022-02-17 | 2022-05-13 | 青岛农业大学 | Preparation and application of dextran magnetic bead simulated BoHV-1 virus particles |
| CN114617982A (en) * | 2022-03-23 | 2022-06-14 | 北京健康启航科技有限公司 | Preparation method of neuroendocrine tumor targeted nanoparticles |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632582A (en) * | 2004-12-27 | 2005-06-29 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Nano immune magnetic particle, preparing process and application |
| WO2007099289A1 (en) * | 2006-02-28 | 2007-09-07 | Medical Research Council | Targeted iron oxide nanoparticles |
| CN101474414A (en) * | 2009-02-06 | 2009-07-08 | 上海师范大学 | Preparation and application of polymer-coated magnetic nanoparticle contrast agent |
| US20140234226A1 (en) * | 2014-05-06 | 2014-08-21 | Morteza Mahmoudi | Plasmonic stable fluorescence superparamagnetic iron oxide nanoparticles and a method of synthesizing the same |
| CN104914241A (en) * | 2014-03-14 | 2015-09-16 | 中国科学院过程工程研究所 | Immunomagnetic bead, preparation method and application thereof |
-
2017
- 2017-03-22 CN CN201710173694.2A patent/CN108287234A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632582A (en) * | 2004-12-27 | 2005-06-29 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Nano immune magnetic particle, preparing process and application |
| WO2007099289A1 (en) * | 2006-02-28 | 2007-09-07 | Medical Research Council | Targeted iron oxide nanoparticles |
| CN101474414A (en) * | 2009-02-06 | 2009-07-08 | 上海师范大学 | Preparation and application of polymer-coated magnetic nanoparticle contrast agent |
| CN104914241A (en) * | 2014-03-14 | 2015-09-16 | 中国科学院过程工程研究所 | Immunomagnetic bead, preparation method and application thereof |
| US20140234226A1 (en) * | 2014-05-06 | 2014-08-21 | Morteza Mahmoudi | Plasmonic stable fluorescence superparamagnetic iron oxide nanoparticles and a method of synthesizing the same |
Non-Patent Citations (3)
| Title |
|---|
| ARIAS SL ET AL.: "Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles.", 《JOURNAL OF VISUALIZED EXPERIMENTS》 * |
| TUDORACHI N ET AL.: "Obtaining of new magnetic nanocomposites based on modified polysaccharide", 《CARBOHYDR POLYM.》 * |
| 牛婉婷 等: "医用葡聚糖包被超顺磁性纳米氧化铁磁珠的制备和表征", 《科技导报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444401A (en) * | 2018-12-12 | 2019-03-08 | 郑州安图生物工程股份有限公司 | A kind of preparation method of magnetic microparticle chemiluminescence product |
| CN114470183A (en) * | 2022-02-17 | 2022-05-13 | 青岛农业大学 | Preparation and application of dextran magnetic bead simulated BoHV-1 virus particles |
| CN114617982A (en) * | 2022-03-23 | 2022-06-14 | 北京健康启航科技有限公司 | Preparation method of neuroendocrine tumor targeted nanoparticles |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ma et al. | Preparation and characterization of monodisperse core–shell Fe3O4@ SiO2 microspheres and its application for magnetic separation of nucleic acids from E. coli BL21 | |
| Nam et al. | Nanoparticles: Synthesis and applications | |
| CN105734043B (en) | Multi-sort cell separation method | |
| Horak et al. | Preparation and properties of magnetic nano‐and microsized particles for biological and environmental separations | |
| Borlido et al. | Magnetic separations in biotechnology | |
| JP6126619B2 (en) | Cell separation method | |
| Ugelstad et al. | Monodisperse magnetic polymer particles: new biochemical and biomedical applications | |
| CN109312293B (en) | Compositions and methods for magnetic levitation separation | |
| Dainiak et al. | Methods in cell separations | |
| CN108287234A (en) | Nano immune magnetic bead and its preparation method and application | |
| Ma et al. | Applications of magnetic materials separation in biological nanomedicine | |
| Chen et al. | Synthesis of immunomagnetic nanoparticles and their application in the separation and purification of CD34+ hematopoietic stem cells | |
| CN106237947A (en) | Magnetic microsphere of high density carboxyl modified and preparation method thereof | |
| CN108176384B (en) | Magnetic nanosphere of grafted arginine polymer brush as well as preparation method and application of magnetic nanosphere | |
| CN102067250B (en) | Magnetic particles | |
| CN106289927A (en) | A kind of from tumor cell supernatant, separate microcapsule bubble and the method for secreting outward body thereof | |
| Zhang et al. | Large-scale immuno-magnetic cell sorting of T cells based on a self-designed high-throughput system for potential clinical application | |
| CN106198963A (en) | A kind of for immunomagnetic beads capturing leukocyte and preparation method thereof | |
| JP2003508073A (en) | Method for modifying selected cells in a magnetic cell separation column | |
| CN107275023B (en) | Golden shell magnetic bead and its preparation method and application | |
| CN112300994B (en) | Nanometer magnetic bead for capturing circulating tumor cells and preparation method and application thereof | |
| CN106366195B (en) | PD-L1 antibody immunomagnetic beads and preparation method thereof | |
| CN104498595B (en) | Cell sorting system based on endonuclease specific recognition | |
| Çakmak et al. | Biofunctionalization of magnetic poly (glycidyl methacrylate) microspheres with protein A: characterization and cellular interactions | |
| CN104450617B (en) | Application of cell sorting system based on specific recognition of endonuclease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180717 |
|
| RJ01 | Rejection of invention patent application after publication |