CN1850966A - Immunomagnetic nano particle cell separator, and its preparing method and use - Google Patents
Immunomagnetic nano particle cell separator, and its preparing method and use Download PDFInfo
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Abstract
The invention relates to an immunity magnetic nanometer particle cell separator that contains inner nuclear layer, N-(2- amino group ethyl)-3-amino group propyl tri-methoxyl group silicane intermediate layer, and antibody and/or ligand outer shell. The invention also relates the method to use the separator.
Description
Technical field
The present invention relates to a kind of nano biological separator, more particularly, the present invention relates to a kind of immunomagnetic nano particle cell separator.The invention still further relates to the Preparation method and use of this separator.
Background technology
As everyone knows, field of nanometer technology is the most popular current scientific and technical research field, nanotechnology is applied to formed emerge science technology-nanometer biotechnology in the bio-science field, be the science of utilizing nanotechnology research and solving the significant problem in the life science, it is just becoming one of current important advanced scientific research field.
Now, the application prospect of magnetic nano-particle in biological technical field is subjected to people's attention day by day.Magnetic nano-particle is through being often used as the separating medium of biological sample magnetic field separation.Separation method with magnetic nano-particle is easy and simple to handle, required equipment is cheap, and velocity of separation is fast simultaneously, helps keeping the biological activity of sample.At present, utilization more and more widely.
Current, in the world hemopoietic stem cell being applied to disease treatment has become the research focus.Especially it is of common occurrence to be applied to the treatment of children malignant tumors and non-malignant tumors and the successful example of leukemia treating, the example that has had 3000 many cases successfully to treat in the world so far, and hemopoietic stem cell CD 34+application the most frequent.
At present, the main source of CD34+ cell is to isolate the CD34+ cell from Cord blood, carries out in-vitro multiplication then.The method of separation of C D34+ cell is a lot of from Cord blood, but all has various shortcoming, can not isolate the CD34+ cell convenient, fast and efficiently.Though people will utilize magnetic systems to separate the hope that is considered as solving this difficult problem, still there is not play-by-play to this class magnetic nano-particle cell separator structure.
Therefore, this area presses for a kind of magnetic nano-particle cell separator of exploitation, so that convenient, fast and isolate the cell that cell surface has the specific antigen mark efficiently, as the CD34+ cell etc.
Summary of the invention
The purpose of this invention is to provide a kind of immunomagnetic nano particle cell separator.
Another object of the present invention provides the method for this immunomagnetic nano particle cell separator of preparation.
A further object of the present invention is that this immunomagnetic nano particle cell separator is applied to separate the cell with specific cells surface marker.
In a first aspect of the present invention, a kind of immunomagnetic nano particle cell separator is provided, it contains following three-decker:
(1) inner nuclear layer that constitutes by the core-shell type magnetic nano particle;
(2) AEAPS[N-(2-the amino-ethyl)-3-TSL 8330 of coating inner nuclear layer] middle layer;
(3) antibody and/or the ligand shell layer in coating middle layer.
In another preference, described core-shell type magnetic nano particle inner nuclear layer comprises kernel and shell.The interior nuclear composition of described inner nuclear layer is selected from: the oxide compound of iron, iron, nickel, nickel-ferro alloy or its combination.Preferably, the interior nuclear composition of described inner nuclear layer is the oxide compound of iron.The outer shell component of described inner nuclear layer is selected from silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination.Preferably, the outer shell component of described inner nuclear layer is a silicon-dioxide.
Wherein, described antibody and/or ligand shell layer are antibody or the parts that is connected with the middle layer by solidifying agent or linking agent (for example ethanol, formaldehyde, glutaraldehyde etc.) or Schiff key.More preferably, described solidifying agent or linking agent are selected from glutaraldehyde.
In another preference, described antibody is anti-CD34 antibody.
In a second aspect of the present invention, a kind of method for preparing immunomagnetic nano particle cell separator is provided, it may further comprise the steps:
(1) in the microemulsion system that contains magnetic nano-particle and inner nuclear layer shell formation agent (as tetraethoxy), by the water-in-oil-type reverse microemulsion process, form the core-shell type magnetic nano particle, described core-shell type magnetic nano particle comprises kernel and shell,
Described magnetic nano-particle is selected from: the oxide compound of iron, iron, nickel, nickel-ferro alloy or its combination;
Described inner nuclear layer shell forms agent and is formed into the shell that branch is selected from down group: silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination;
(2) use N-(2-amino-ethyl)-3-TSL 8330 that the core-shell type magnetic nano particle surface of step (1) is modified, obtained modifying amino magnetic nano-particle;
(3) use antibody or part to the modification of step (2) surface of amino magnetic nano-particle modify, form immunomagnetic nano particle cell separator.
Wherein, in the described microemulsion system TritonX-100, n-hexyl alcohol, hexanaphthene in 1: (1-3): ratio uniform mixing (4-6).
In a third aspect of the present invention, the purposes of immunomagnetic nano particle cell separator of the present invention is provided, it is used to isolated cell, and the surface of described cell has the antigen with described antibodies, perhaps with described part bonded acceptor.
In a fourth aspect of the present invention, a kind of method of isolated cell is provided, comprise step: this immunomagnetic nano particle cell separator is contacted with the solution that contains target cell, form the mixture of described immunomagnetic nano particle cell separator and target cell, isolate described mixture under the guiding of outside magnetic field then.
Description of drawings
Fig. 1 is the surface-enhanced Raman effects figure that has modified amino magnetic nano-particle, shows that amino is modified at the surface of magnetic nano-particle, and the amino that is modified at the magnetic nano particle sub-surface has good Raman reinforcing effect in the substrate of silver.
Fig. 2 utilizes immune magnetic nano particle to carry out the design sketch of cellular segregation.2 times separating resulting all shows, this cell separator is separation of C D34+ hemopoietic stem cell effectively, and good separating effect is arranged.
Fig. 3 A and 3B are the figure that cultivates back propagation by the cell that cell separator is separated.Wherein Fig. 3 A is cell cultures observed colony after ten days; Fig. 3 B is an observed colony behind the cell cultures fortnight.This shows that the cell of separating still has good biological activity.
Embodiment
The present inventor is through extensive and deep research, invented in the finishing of magnetic nano-particle and can discern and proteinaceous substancess such as binding antibody or part, remove the acquisition target cell with this magnetic nano-particle then, carry out isolating technology under the effect of outside magnetic field again.
The method for preparing immunomagnetic nano particle cell separator of the present invention may further comprise the steps:
(1) preparation magnetic nano-particle
Prepare FeSO respectively with redistilled water
4.7H
2O and FeCl
3.6H
2The mixing solutions of O and NaOH solution.Fe in the mixing solutions of molysite
2+Ionic concentration is 0.1-0.2mol/l, Fe
3+Ionic concentration is 0.1-0.3mol/l, and the concentration of NaOH solution is 2-3mol/l.Under vigorous stirring be that half NaOH solution of mixing salt solution volume is added drop-wise in the mixing salt solution lentamente with volume.At 40 ℃ of-60 ℃ of following ageing 12h, with redistilled water with sediment undergoes washing for several times, dry 24h under 40 ℃-80 ℃ condition again after the filtration promptly gets product after grinding in agate mortar with resulting solid precipitation.
(2) silicon-dioxide is at γ-Fe
2O
3The modification on surface
With TritonX-100, n-hexyl alcohol, hexanaphthene in 1: (1-3): ratio uniform mixing (4-6) forms the microemulsion system of transparent and stable.Place ultrasonic wave to handle 30-60 minute above-mentioned microemulsion system, again to the γ-Fe that wherein adds 0.01-1g
2O
3(magnetic nano-particle) takes out upper strata liquid with ultrasonication and pours in the three-necked flask after 3 minutes, stir and made it even in 30-60 minute.Get the certain density strong aqua of 1ml with the dilution of 2ml redistilled water, it is slowly joined in the microemulsion of continuous stirring, continue stirring and ammoniacal liquor was dispersed in the microemulsion in 10-60 minute.After 1 hour, in microemulsion, drip the tetraethoxy (the inner nuclear layer shell forms agent) of 1-5ml, constantly stirred 10 hours simultaneously, and the temperature of system is remained between 15-40 ℃.In system, add acetone and make particle precipitation, perhaps the system standing over night is made the particle natural sedimentation, use the ethanol wash particle.Particle after cleaning placed under 300-700 ℃ the condition calcination 1-4 hour, and collected the core-shell type magnetic nano particle.
(3) with N-(2-amino-ethyl)-3-TSL 8330 (AEAPS) modified magnetic nano particles surface
(methyl alcohol: the volume ratio of glycerol is 2: 3-2: 1), use ultrasonication 20-60 minute to get the methyl alcohol of 15mg magnetic nano-particle adding 30-50ml and the mixed solution of glycerol; Take by weighing 0.01-1gAEAPS (N-(2-amino-ethyl)-3-TSL 8330), use ultrasonication 10-60 minute; These two kinds of solution are mixed, and reaction is 1-6 hour under 10-95 ℃ condition, takes out particle then and uses washed with methanol 3 times, then 100-300 ℃ of vacuum-drying, collects particle (FSM).
(4) antibody or part are in the modification of magnetic nano particle sub-surface
Get the particle of preparation in the step (3), add in the buffer solution system that pH is 7.0-8.0 (as phosphate buffer), add consumption, form mixing solutions for to make the concentration of linking agent (or solidifying agent) in system be about 0.5%-2%.Described mixing solutions is used ultrasonication 30-60 minute.Then with phosphate buffered saline buffer washing about 1-5 time (more preferably 2-3 time).
Get a certain amount of antibody or part (though be not particularly limited with the weight or the molar concentration rate of magnetic nano-particle, usually with 1: 6-1: 3 is good), join in the mixing solutions of above-mentioned particle.Reaction is 1-6 hour under 4-40 ℃ temperature of reaction, thus with the COOH on antibody or the part under the effect of fixing agent with the middle layer on amino reaction, make antibody or part be directly connected in the middle layer by the Schiff key.After reaction finished, the described particle of magnetic resolution was with about 2-3 time of phosphate buffered saline buffer washing.It is standby that the immunomagnetic nano particle cell separator that obtains like this can place phosphate buffered saline buffer to preserve.
Be applicable to that antibody of the present invention or part have no particular limits.Can be any antibody or part at antigen on the cell surface or acceptor.For example, the antigenic antibody of anti-CD34.
The interior nuclear composition of core-shell type magnetic nano particle inner nuclear layer also can comprise the alloy of iron, nickel (Ni) or nickel (Ni) and iron (Fe) etc. except the oxide compound of iron.
The outer shell component of core-shell type magnetic nano particle inner nuclear layer also has organic integuments such as agarose, olefin polymer, polyacrylonitrile, epoxy compounds except inorganic integuments such as silicon-dioxide.For selected outer shell component, can select for use suitable inner nuclear layer shell to form agent according to prior art.For example when outer shell component is silicon-dioxide, can select for use tetraethoxy or other suitable inner nuclear layer shell to form agent.
Major advantage of the present invention is:
(1) magnetic nano-particle of Shi Yonging is a monodispersity, does not produce agglomeration, and the shape of magnetic nano-particle and diameter are easy to control;
(2) antibody or the part that can discern target cell is connected with magnetic nano-particle by chemical bond, and stability is good, difficult drop-off;
(3) the inventive method is easy and efficient.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The preparation method of the inner nuclear layer of magnetic nano-particle
Adopt improved chemical coprecipitation to prepare the inner nuclear layer of magnetic particle, concrete grammar is as follows: prepare FeSO respectively with redistilled water
4.7H
2O and FeCl
3.6H
2The mixing solutions of O and NaOH solution.Fe in the mixing solutions of molysite
2+Ionic concentration is 0.1-0.2mol/l, Fe
3+Ionic concentration is 0.1-0.3mol/l, and the concentration of NaOH solution is 2-3mol/l.Under vigorous stirring be that half NaOH solution of mixing salt solution volume is added drop-wise in the mixing salt solution lentamente with volume.At 40 ℃ of-60 ℃ of ageing 12h, with redistilled water with sediment undergoes washing for several times, dry 24h under 40 ℃-80 ℃ condition again after the filtration promptly gets product after grinding in agate mortar with resulting solid precipitation.
The preparation method of hud typed nucleic acid magnetic nano-particle
With TritonX-100, n-hexyl alcohol, hexanaphthene ratio uniform mixing, form the microemulsion system of transparent and stable in 1: 2: 5.Place ultrasonic wave to handle 30-60 minute above-mentioned microemulsion system, again to the γ-Fe that wherein adds 0.5g
2O
3, take out upper strata liquid after 6 minutes with ultrasonication and pour in the three-necked flask, stir and made it even in 30 minutes.Get the certain density strong aqua of 1ml with the dilution of 2ml redistilled water, after 30 minutes it is slowly joined in the microemulsion of continuous stirring, continue stirring and ammoniacal liquor was dispersed in the microemulsion in 30 minutes.After 1 hour, in microemulsion, drip the tetraethoxy of 1-3ml, constantly stirred 10 hours simultaneously, and the temperature of system is remained between 15-30 ℃.In system, add acetone and make particle precipitation, perhaps the system standing over night is made the particle natural sedimentation, use the ethanol wash particle.Particle after cleaning placed under 400-700 ℃ the condition, calcination 1-4 hour, collect particle.
Embodiment 3
The preparation of immunomagnetic nano particle cell separator
(a) with hydride modified magnetic nano particle sub-surface
Get the magnetic nano-particle that makes among the 20mg embodiment 2, in the methyl alcohol of adding 50ml and the mixed solution of 5: 3 composition of glycerol, use ultrasonication 20-60 minute; Take by weighing 3mlAEAPS, use ultrasonication 10-60 minute; These two kinds of solution are mixed, and reaction is 5 hours under 60 ℃ condition, takes out particle then and uses washed with methanol 3 times, then 40-80 ℃ of vacuum-drying 2 hours, collects particle.
As can be seen from Figure 1, the amino surface of being modified magnetic nano-particle, and good Raman reinforcing effect is arranged.
(b) at the finishing antibody of magnetic nano-particle
Get the particle that 1-3mg as above prepares, add in the phosphate buffer that pH is 7.0-8.0, adding consumption is 50-200 μ l linking agent (glutaraldehyde), forms mixing solutions.Described mixing solutions is used ultrasonication 10-30 minute.Then with phosphate buffered saline buffer washing 2-3 time.
Get the anti-CD34 antibody (available from Biomeda company) of about 3 micrograms, join in the mixing solutions of the above-mentioned particle that contains about 2ml.Reaction is 3 hours under 2-10 ℃ temperature of reaction, thereby COOH on the antibody and the amino on the middle layer are reacted under the effect of linking agent, makes antibody be directly connected in the middle layer by the Schiff key.After reaction finished, magnetic resolution went out described particle, with phosphate buffered saline buffer washing 3 times.The immunomagnetic nano particle cell separator that obtains like this be placed in the phosphate buffered saline buffer preserve standby.
Embodiment 4
The preparation of immunomagnetic nano particle cell separator
(a) with hydride modified magnetic nano particle sub-surface
Get the magnetic nano-particle that makes among the 10-20mg embodiment 2, in the mixed solution that 1: 1 the methyl alcohol of adding 30ml and glycerol are formed, use ultrasonication 20-60 minute; Take by weighing 2mlAEAPS, use ultrasonication 10-60 minute; These two kinds of solution are mixed, and reaction is 2-4 hour under 60-85 ℃ condition, takes out particle then and uses washed with methanol 3 times, then 60 ℃ of vacuum-dryings 2 hours, collects particle.
(b) antibody is to the modification of magnetic nano-particle
Get the particle that 0.5mg as above prepares, add in the phosphate buffer that pH is 7.0-8.0, adding consumption is 50-100 μ l linking agent (glutaraldehyde), forms mixing solutions.Described mixing solutions is used ultrasonication 30-60 minute.Then with phosphate buffered saline buffer washing 2-3 time.
Get the anti-CD34+ monoclonal antibody (available from Biomeda company) of about 1.5 micrograms, join in the mixing solutions of the above-mentioned particle that contains about 0.5mg.Reaction is 3 hours under 2-10 ℃ temperature of reaction, thereby COOH on the antibody and the amino on the middle layer are reacted under the effect of linking agent, makes antibody be directly connected in the middle layer by Schiff's base.After reaction finished, magnetic resolution went out described particle, with phosphate buffered saline buffer washing 3 times.The immunomagnetic nano particle cell separator that obtains like this be placed in the phosphate buffered saline buffer preserve standby.
Embodiment 5
The preparation of immunomagnetic nano particle cell separator
(a) with hydride modified magnetic nano particle sub-surface
Get the magnetic nano-particle that makes among the 30mg embodiment 2, in the mixed solution that 5: 3 the methyl alcohol of adding 50ml and glycerol are formed, use ultrasonication 20-60 minute; Measure 3ml AEAPS, use ultrasonication 10-60 minute; These two kinds of solution are mixed, and reaction is 3 hours under 50-85 ℃ condition, takes out particle then and uses washed with methanol 3 times, then 40-80 ℃ of vacuum-drying 2 hours, collects particle.
(b) antibody is to the modification of magnetic nano-particle
Get the particle that 1mg as above prepares, add in the phosphate buffer that pH is 7.0-8.0, adding consumption is 50-150 μ l linking agent (glutaraldehyde), forms mixing solutions.Described mixing solutions is used ultrasonication 30-60 minute.Then with phosphate buffered saline buffer washing 2-3 time.
Get the anti-CD34 antibody (available from Biomeda company) of about 3 micrograms, join in the mixing solutions of the above-mentioned particle that contains about 2mg.Reaction is 3 hours under 2-10 ℃ temperature of reaction, thereby COOH on the antibody and the amino on the middle layer are reacted under the effect of linking agent, makes antibody be directly connected in the middle layer by forming the Schiff key.After reaction finished, magnetic resolution went out described particle, with phosphate buffered saline buffer washing 3 times.The immunomagnetic nano particle cell separator that obtains like this be placed in the phosphate buffered saline buffer preserve standby.
Embodiment 6
Separate stem cells
Get the immunomagnetic nano particle cell separator 100-200 μ l (0.5mg/ml) that makes among the embodiment 3-5, add in the celliferous mixing solutions of 0.5ml, placed 0.25-0.5 hour down at 4 ± 2 ℃.Wherein, in described celliferous nutrient solution, contain and have an appointment 3 * 10
4Individual target cell and 9 * 10
5Individual other irrelevant mononuclearcells.
Under the effect in external magnetic field, collect magnetic nano-particle, remove clear liquid.Clean magnetic nano-particle three times with the RPMI-1640 nutrient solution.Magnetic nano-particle is suspended in the cell culture fluid, with flow cytometer or microscopic examination separating resulting.
The result: the hud typed nucleic acid magnetic nano-particle that embodiment 3-5 makes is a monodispersity, can not produce agglomeration, and its shape and diameter are easy to control.In addition, can discern CD34+ cell (target cell), and the CD34+ cell is good in conjunction with stability, difficult drop-off, so separating effect is very good.
As can be seen from Figure 2 the separating effect of immunomagnetic beads has reached more satisfactory result's (per-cent of CD34+ cell is 0.5-4% in the Cord blood).
The cell of as can be seen from Figure 3 separating still has very high multiplication capacity.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a magnetic nano-particle cell separator is characterized in that, it contains following three-decker:
(1) inner nuclear layer that constitutes by the core-shell type magnetic nano particle;
(2) middle layer of coating inner nuclear layer;
(3) antibody and/or the ligand shell layer in coating middle layer.
2. magnetic nano-particle cell separator according to claim 1 is characterized in that, core-shell type magnetic nano particle inner nuclear layer comprises kernel and shell.
3. magnetic nano-particle cell separator according to claim 2 is characterized in that, the interior nuclear composition of described inner nuclear layer is selected from: the oxide compound of iron, iron, nickel, nickel-ferro alloy or its combination.
4. magnetic nano-particle cell separator according to claim 3 is characterized in that, the interior nuclear composition of described inner nuclear layer is the oxide compound of iron.
5. magnetic nano-particle cell separator according to claim 2 is characterized in that, the outer shell component of described inner nuclear layer is selected from silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination.
6. magnetic nano-particle cell separator according to claim 5 is characterized in that, the outer shell component of described inner nuclear layer is a silicon-dioxide.
7. magnetic nano-particle cell separator according to claim 1 is characterized in that, described antibody and/or ligand shell layer are antibody or the parts that is connected with the middle layer by the Schiff key.
8. method for preparing the described magnetic nano-particle cell separator of claim 1 is characterized in that it may further comprise the steps:
(1) in the microemulsion system that contains magnetic nano-particle and inner nuclear layer shell formation agent, by the water-in-oil-type reverse microemulsion process, form the core-shell type magnetic nano particle, described core-shell type magnetic nano particle comprises kernel and shell,
Described magnetic nano-particle is selected from: the oxide compound of iron, iron, nickel, nickel-ferro alloy or its combination;
Described inner nuclear layer shell forms agent and is formed into the shell that branch is selected from down group: silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination;
(2) use N-(2-amino-ethyl)-3-TSL 8330 that the core-shell type magnetic nano particle surface of step (1) is modified, obtained modifying amino magnetic nano-particle;
(3) with antibody or part to the modification of step (2) surface of amino magnetic nano-particle modify, form immunomagnetic nano particle cell separator.
9. method according to claim 8 is characterized in that, TritonX-100, n-hexyl alcohol, hexanaphthene are in 1 in the described microemulsion system: 1-3: the ratio uniform mixing of 4-6.
10. the purposes of magnetic nano-particle cell separator as claimed in claim 1 is characterized in that, is used for isolated cell, and the surface of described cell has the antigen with described antibodies, perhaps with described part bonded acceptor.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014075629A1 (en) * | 2012-11-15 | 2014-05-22 | Hong Kong Baptist University | Method of extracting neural stem cells using nanoparticles |
| US10383959B2 (en) | 2012-11-15 | 2019-08-20 | Hong Kong Baptist University | Dual-mode contrast agent and uses thereof in real-time monitoring and harvesting of neural stem cells |
-
2005
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014075629A1 (en) * | 2012-11-15 | 2014-05-22 | Hong Kong Baptist University | Method of extracting neural stem cells using nanoparticles |
| CN104837986A (en) * | 2012-11-15 | 2015-08-12 | 香港浸会大学 | Method of extracting neural stem cells using nanoparticles |
| EP2920298A4 (en) * | 2012-11-15 | 2016-07-06 | Univ Hong Kong Baptist | Method of extracting neural stem cells using nanoparticles |
| CN104837986B (en) * | 2012-11-15 | 2018-12-07 | 香港浸会大学 | The method for extracting neural stem cell using nanoparticle |
| US10383959B2 (en) | 2012-11-15 | 2019-08-20 | Hong Kong Baptist University | Dual-mode contrast agent and uses thereof in real-time monitoring and harvesting of neural stem cells |
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Assignee: Beijing Hanshi United Biological Technology Co., Ltd. Assignor: Shanghai Normal University Contract record no.: 2011110000176 Denomination of invention: Immunomagnetic nano particle cell separator, and its preparing method and use Granted publication date: 20090722 License type: Exclusive License Open date: 20061025 Record date: 20111008 |
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