CN109136164A - A method of extracellular vesica is separated based on LBL method modification heparin - Google Patents
A method of extracellular vesica is separated based on LBL method modification heparin Download PDFInfo
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- CN109136164A CN109136164A CN201811054860.8A CN201811054860A CN109136164A CN 109136164 A CN109136164 A CN 109136164A CN 201811054860 A CN201811054860 A CN 201811054860A CN 109136164 A CN109136164 A CN 109136164A
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Abstract
The invention discloses a kind of methods for separating extracellular vesica based on LBL method modification heparin.The specific steps of which are as follows: the method that (1) is modified by LBL layer by layer first successively alternately modifies heparin and diallyl dimethyl ammoniumchloride PDADMAC with the principle of Electrostatic Absorption on several positively charged nanospheres；Decorative layer is arranged according to the sequence of heparin, PDADMAC, heparin from inside to outside, and the number of plies of decorative layer is the odd-level more than or equal to three layers；(2) nanosphere after modification is added to after reacting 5min-120min in the body fluid containing extracellular vesica, centrifugation or magnetic suck collect nanosphere；(3) sodium chloride solution reaction is added in the nanosphere of collection, the extracellular vesica for being adsorbed on nanometer ball surface is eluted in sodium chloride solution, realizes the separation of extracellular vesica in body fluid.The method of the present invention is simple and reliable, cost is extremely low, flexibility ratio is big.
Description
Technical field
The invention belongs to field of biomedicine technology, it is related to a kind of separating extracellular vesica based on LBL method modification heparin
Method.
Background technique
Extracellular vesica generally includes excretion body, microcapsule bubble and apoptotic body etc., carries various albumen, nucleic acid, or even complete
The DNA of genome, can be used as courier, pass through the conduction of the signal between the approach mediated cell such as blood, urine, milk, saliva.It grinds
Study carefully the generation, development and transfer for showing that extracellular vesica has mediated tumour, be able to reflect the relevant information of tumour, is a kind of
Potential tumor cells marker.Extracellular vesica isolates and purifies the main bottleneck for being the research of excretion body and applying.Current
Extracting method include: supercentrifugation based on density, based on size be separated by filtration method, the immunity enrichment method based on antibody,
The PEG precipitation method etc. based on similar isolated viral.For supercentrifugation, equipment and consumptive material are expensive, take a long time, purity
It is low.It is separated by filtration method easily to cause to block, material consumption is larger, takes a long time.It is extracellular that high-purity can be obtained in immunity enrichment method
Vesica, but Antibody preparation is complicated, it is expensive, it is unstable between batch.The PEG precipitation method can be inexpensive quickly isolated thin
Extracellular vesica, but purity is low, and protein contamination is serious.
2015 it was discovered by researchers that can by the Ago-Gel nanosphere with heparin prepared by covalent modification methods
With vesica outside special combination cell, and can be by under the combining extracellular vesica elution of high concentration sodium chloride solution
Come, to obtain the higher extracellular vesica of purity.The method of this extracellular vesica of separation and concentration is at low cost, and speed is fast, is
The research and application of extracellular vesica provide strong support.But there is also certain problems, first agarose for this method
For gel nano sheet as porous material, adherent cell outer vesica while, can also adsorb a certain amount of nucleic acid or protein etc.
Impurity, impurity can also elute together when elution, influence purity.Secondly, heparin is to be modified by covalent in agar
On sugared gel nano, modification needs certain chemical reaction, increases separation costs.
The method of LBL self-assembly LBL modification, is two kinds of macromolecular complex with opposite-sign matter or opposite hydrophilic property
Matter, by the method for layer-by-layer, by the modification to substrate of macromolecular substances in layer, to realize macromolecular
Modification to surface.The modification that specific molecular inexpensive, quickly can be carried out to surface, desired property is obtained on surface.
Summary of the invention
For overcome the deficiencies in the prior art, the purpose of the present invention is to provide one kind based on the modification heparin separation of LBL method
The method of extracellular vesica.The method that the present invention separates extracellular vesica is simple and reliable, cost is extremely low, flexibility ratio is big, separation is fast
Degree is fast, good separating effect.
Technical solution of the present invention is specifically described as follows.
A method of extracellular vesica is separated based on LBL method modification heparin, the specific steps are as follows:
(1) method modified layer by layer by LBL first, with the principle of Electrostatic Absorption, on several positively charged nanospheres
Successively alternately modify heparin and diallyl dimethyl ammoniumchloride PDADMAC；Decorative layer from inside to outside according to heparin,
PDADMAC, heparin sequence arranged, the number of plies of decorative layer is the odd-level more than or equal to three layers；
(2) nanosphere after modification is added to after reacting 5min-120min in the body fluid containing extracellular vesica, centrifugation
Or magnetic suck collects nanosphere；
(3) 2.0~2.5mol/L sodium chloride solution is added in the nanosphere of collection and reacts 10~20min, be adsorbed on and receive
The extracellular vesica of rice ball surface is eluted in sodium chloride solution, is discarded nanosphere by centrifugation or magnetic suck, is realized body fluid
In extracellular vesica separation.
In the present invention, in step (1), positively charged nanosphere is by polystyrene, ferroso-ferric oxide, silicon or silica
The nanosphere of material is obtained by amination or silanization treatment.
In the present invention, in step (1), the number of positively charged nanosphere is 50,000-100 ten thousand, partial size 50 nanometers~
Between 10 microns.
In the present invention, in step (1), modified on positively charged nanosphere using the aqueous solution or PBS solution of heparin
Heparin, modification time are 1~60min；It is modified on positively charged nanosphere using the aqueous solution or PBS solution of PDADMAC
PDADMAC, modification time are 1~60min.
In the present invention, in step (1), the aqueous solution of heparin or the concentration of PBS solution are 0.5mg/mL-20mg/mL,
The aqueous solution of PDADMAC or the concentration of PBS solution are 0.5mg/mL-20mg/mL.
In the present invention, in step (2), body fluid is any in blood plasma, urine, milk, bile or saliva.
Compared to the prior art, the beneficial effects of the present invention are the method for the present invention simple process, least situation lower
It needs the bead that will have positive electricity to modify three layers of polymer substance by the effect of electrostatic force, can realize to have and specially adsorb
The function of extracellular vesica, wherein heparin and PDADMAC are common high molecular material, cheap, are easy to get, and from
There is no toxicity, more environmental protection for modification process and material itself.Bead after modification can be mixed directly with body fluid,
To realizing the low cost of extracellular vesica, quickly and efficiently separate, isolated extracellular vesica has high-purity, can be with
Directly carry out the experiment and analysis of subsequent step, and since bead is by being centrifuged or the method for magnetic absorption facilitates separation, because
This can be relatively easy to the automation for realizing extracellular vesica separation and content analysis.
Detailed description of the invention
Fig. 1 is nanosphere modification schematic diagram.
Fig. 2 is extracellular vesica seperated schematic diagram.
Fig. 3 is that the time is modified in embodiment 1 to the influence diagram for extracting Evs.
Fig. 4 is absorption front and back Evs partial size and concentration variation diagram in embodiment 1.
Fig. 5 is the influence diagram modifying the number of plies in embodiment 2 and extracting to EVs.
Fig. 6 is that bead non-specific adsorption DNA and haemocyanin test chart are modified in embodiment 3.
Fig. 7 is the potential change figure in embodiment 4 between nanosphere different modifying layer.
Fig. 8 is that nanosphere is schemed by the TEM of LBL modification front and back in embodiment 5；(a) before modifying, after (b) modifying.
Fig. 9 is the TEM figure modified after the outer vesica of nanosphere adherent cell in embodiment 5.
Figure 10 is the TEM figure of the isolated extracellular vesica of embodiment 6.
Specific embodiment
The present invention passes through the method that LBL is modified layer by layer, with the principle of Electrostatic Absorption, by the heparin with negative electricity and with just
Diallyl dimethyl ammoniumchloride (PDADMAC) modification of electricity is as shown in Figure 1 on the nanosphere with positive electricity.Wherein nanometer
The material of ball can modify the nanosphere of positive electricity for polystyrene, ferroso-ferric oxide, silicon, silica etc..With positive electricity
Nanosphere size be 50 nanometers to 10 microns.The positive charged group of modification includes amino, silanization etc..Heparin is used when modification
Aqueous solution or PBS solution, heparin concentration 0.5mg/ml-20mg/ml.PDADMAC aqueous solution or PBS solution are used when modification,
PDADMAC concentration is 0.5mg/ml-20mg/ml.The nanosphere number used when modification is 50,000-100 ten thousand.To nanosphere
It is modified to one layer of heparin, one layer of PDADMAC, one layer of heparin, is successively modified, the modification number of plies is not limited to three layers, but decorative layer
Number is singular layer, such as 3,5,7, according to the actual situation depending on, outermost layer keeps heparin.The modification number of plies is more, and the structure of LBL is more steady
It is fixed, but can then be greatly prolonged the time required to corresponding modification.Therefore, if being considered from disengaging time, if necessary to quickly divide
When from extracellular vesica, then the modification of the number of plies can be reduced, if sample will affect LBL structure, it is contemplated that increasing
The number of plies is modified, come the stabilization of LBL bead when guaranteeing separation.Nanosphere is added to the body containing extracellular vesica after the completion of modification
In liquid, body fluid includes blood plasma, urine, milk, bile, saliva etc..After acting on 5min-120min, centrifugation or magnetic suck are collected
Nanosphere.At this point, extracellular vesica is then adsorbed on a nanometer ball surface.After separating nanosphere, sodium chloride solution effect is added, it can be with
It is eluted to the extracellular vesica for being adsorbed on nanometer ball surface in solution.Nanosphere is discarded, the outer vesica of final cell realizes separation,
Seperated schematic diagram is as shown in Figure 2.It describes in detail combined with specific embodiments below to technical solution of the present invention.
Embodiment 1
The influence that the modification time extracts EVs
1, take the amido modified ferroferric oxide magnetic nano ball with positive electricity of 3 microns of 1,500,000 diameters to 5ml centrifuge tube
In, it is divided into three parts with 2ml centrifuge tube, is mixed respectively with 1ml heparin solution (2mg/ml), acts on 10min, 30min, 1h.
2, magnetic suck, collection nanosphere, 1ml pure water are washed three times respectively, and magnetic is attached to centrifuge tube bottom, discards liquid.
3,1mlPDADMAC aqueous solution (2mg/ml) is added into centrifuge tube respectively, acts on 10min, 30min, 1h.Respectively
It is washed three times with 1ml pure water, magnetic is attached to centrifuge tube bottom, discards liquid.
4,1ml heparin solution (2mg/ml) mixing is added into centrifuge tube respectively, acts on 10min, 30min, 1h.Respectively
It is washed three times with 1ml pure water, magnetic is attached to centrifuge tube bottom, discards liquid.
5, the culture solution after taking the culture 12h of 200mlPC-12 cell strain.4 DEG C of 100000g are centrifuged 70min, isolated
Extracellular vesica precipitating, discards supernatant, and 2ml PBS buffer solution is added, the extracellular vesica of precipitating is resuspended.
6, the bead of different modifying time and the light maintenance being not decorated are added to the extracellular vesica of resuspension of 0.5ml respectively
In, adsorb 1h.
7, magnet adsorbing separation bead is collected into new centrifuge tube to centrifuge tube tube bottom, supernatant.Bead is washed with PBS
3 times, and cleaning solution is collected, final bead of collecting is to centrifuge tube bottom.
8,1ml 2M sodium chloride solution, 4 DEG C of effect 12h, to the cell being adsorbed on bead are added to centrifuge tube bottom bead
Outer vesica is eluted, and bead is discarded, and collects supernatant.
9, respectively in the three kinds of solution nanosight test solution collected in above-mentioned experiment extracellular vesica it is dense
Degree, and calculate the ratio for accounting for the outer vesica concentration of initial cell.As a result as shown in Figure 3.Experiment discovery, modification 30min's or more is small
Ball is 60% or more to the separative efficiency of extracellular vesica, and little with the difference of 1h.Therefore, subsequent experimental is using modification
The method of 30min is tested.To modification 30min bead, separate EVs partial size and concentration variation counted as shown in figure 4,
The EVs particle diameter distribution eluted remains as~120nm, meets the literature values of EVs partial size.
Embodiment 2
The influence that the modification number of plies extracts EVs
1, take the amido modified ferroferric oxide magnetic nano ball with positive electricity of 3 microns of 1,500,000 diameters to 5ml centrifuge tube
In, it is divided into three parts with 2ml centrifuge tube, uses 1ml heparin solution (3mg/ml) and 1mlPDADMAC aqueous solution (3mg/ml) respectively
30min alternately is modified to bead.Obtain 1 layer of bead@HA of modification, the bead@HA of 3 layers of modification PDADMAC HA, modify 5 layers
Bead@HA PDADMAC HA PDADMAC HA.
2, the culture solution after taking the culture 12h of 200mlPC-12 cell strain.4 DEG C of 100000g are centrifuged 70min, isolated
Extracellular vesica precipitating, discards supernatant, and 2ml PBS buffer solution is added, the extracellular vesica of precipitating is resuspended.
3, the bead of the different modifying number of plies and the light maintenance being not decorated are added to the extracellular vesica of resuspension of 0.5ml respectively
In, adsorb 1h.
4, magnet adsorbing separation bead is collected into new centrifuge tube to centrifuge tube tube bottom, supernatant.Bead is washed with PBS
3 times, and cleaning solution is collected, final bead of collecting is to centrifuge tube bottom.
5,1ml 2M sodium chloride solution, 4 DEG C of effect 12h, to the cell being adsorbed on bead are added to centrifuge tube bottom bead
Outer vesica is eluted, and bead is discarded, and collects supernatant.
6, respectively in the three kinds of solution nanosight test solution collected in above-mentioned experiment extracellular vesica it is dense
Degree, and calculate the ratio for accounting for the outer vesica concentration of initial cell.As a result as shown in Figure 5.Experiment discovery, the bead of 3 layers of modification or more
To the separative efficiency of extracellular vesica close to 60%, and it is little with 5 layers of difference.Therefore, subsequent experimental decorative layer number according to
Experiment goes to ask variation.
Embodiment 3
Modify bead non-specific adsorption DNA and haemocyanin test
1, take the amido modified ferroferric oxide magnetic nano ball with positive electricity of 2 microns of 300,000 diameters to 2ml centrifuge tube
In, it is mixed with 1ml heparin PBS solution (5mg/ml), acts on 15min.
2,15000 turns/min is centrifuged, and collects nanosphere, and 1mlPBS is washed three times, 15000 turns/min centrifugation to centrifuge tube bottom,
Discard liquid.
3, the PBS solution (5mg/ml) of 1mlPDADMAC is added into centrifuge tube, acts on 30min.1mlPBS is washed three times,
15000 turns/min is centrifuged to centrifuge tube bottom, discards liquid.
4,1ml heparin PBS solution (5mg/ml) mixing is added into centrifuge tube, acts on 30min.1mlPBS is washed three times,
15000 turns/min is centrifuged to centrifuge tube bottom, discards liquid.It is spare.
5, the BSA solution of 70mg/ml is prepared, haemocyanin environment is simulated.Prepare the double stranded DNA solutions of 0.08mg/ml, mould
CfDNA in quasi- blood plasma.1ml and modification bead effect 1h are taken respectively.
6, after magnetic absorption bead to centrifugation bottom of the tube, the BSA of supernatant and DNA is carried out quantitative (Fig. 6) respectively, in discovery
BSA and DNA in clear are reduced there is no apparent.Illustrate that with protein and DNA non-specific adsorption will not occur for bead.
7, it discarding supernatant, after adding PBS to be resuspended the bead for being deposited to tube bottom, surveys Zeta potential, experimental result is shown, with
After BSA and DNA interaction, there is no significantly changing for the surface electrification of bead.Illustrate bead will not with protein and
Non-specific adsorption occurs for DNA.
Embodiment 4
1, take the amido modified ferroferric oxide magnetic nano ball with positive electricity of 2 microns of 300,000 diameters to 2ml centrifuge tube
In, it is mixed with 1ml heparin solution (5mg/ml), acts on 30min.
2, magnetic suck collects nanosphere, and 1ml pure water is washed three times, and magnetic is attached to centrifuge tube bottom, discards liquid.
3,1mlPDADMAC aqueous solution (5mg/ml) is added into centrifuge tube, acts on 30min.1ml pure water is washed three times, magnetic
It is attached to centrifuge tube bottom, discards liquid.
4,1ml heparin solution (5mg/ml) mixing is added into centrifuge tube, acts on 30min.1ml pure water is washed three times, magnetic
It is adsorbed onto centrifuge tube bottom, discards liquid.
5,1mlPDADMAC aqueous solution (5mg/ml) is added into centrifuge tube, acts on 30min.1ml pure water is washed three times, magnetic
It is attached to centrifuge tube bottom, discards liquid.
6,1ml heparin solution (5mg/ml) mixing is added into centrifuge tube, acts on 30min.1ml pure water is washed three times, magnetic
It is adsorbed onto centrifuge tube bottom, discards liquid.It is spare.The nanosphere surface potential variation of five layers of modification is as shown in Figure 7.
7,1ml blood is taken, 2600g is centrifuged 15min, removes cell and cell fragment in body fluid.
8, the blood plasma after centrifugation is added in the modification nanosphere that step 6 precipitates, room temperature acts on 60min.
9, nanosphere is sunken to centrifuge tube bottom by magnetic suck, discards liquid.
10,1ml 2M NaCl solution is added, after acting on 15min, nanosphere is sunken to centrifuge tube bottom by magnetic suck, and suction contains
There is the liquid of extracellular vesica, -20 DEG C of preservations are spare.
Embodiment 5
1, take polystyrene nanospheres of 1,000,000 diameters, the 1 micron of silylation modification with positive electricity into 2ml centrifuge tube,
It is mixed with 1ml heparin PBS solution (1mg/ml), acts on 15min.
2,15000 turns/min is centrifuged, and collects nanosphere, and 1mlPBS is washed three times, 15000 turns/min centrifugation to centrifuge tube bottom,
Discard liquid.
3, the PBS solution (1mg/ml) of 1mlPDADMAC is added into centrifuge tube, acts on 30min.1mlPBS is washed three times,
15000 turns/min is centrifuged to centrifuge tube bottom, discards liquid.
4,1ml heparin PBS solution (1mg/ml) mixing is added into centrifuge tube, acts on 30min.1mlPBS is washed three times,
15000 turns/min is centrifuged to centrifuge tube bottom, discards liquid.It is spare.The TEM of modification front and back schemes as shown in figure 8, can significantly see
To by negative staining, nanometer ball surface is formd and nanosphere contrast difference itself.Show to modify successfully.
5,10ml urine is taken, 2600g is centrifuged 15min, removes cell and cell fragment in body fluid.
6, the urine after centrifugation is added in the modification nanosphere that step 4 precipitates, room temperature acts on 60min.
7, nanosphere is sunken to centrifuge tube bottom by 15000 turns/min centrifugation, discards liquid.Nanometer after the outer vesica of adherent cell
Ball is as shown in Figure 9.
8,1ml 2M NaCl solution is added, after acting on 15min, nanosphere is sunken to centrifuge tube by 15000 turns/min centrifugation
Bottom, is sucked out the liquid for containing extracellular vesica, and -20 DEG C of preservations are spare.
Embodiment 6
1, take the amido modified ferroferric oxide magnetic nano ball with positive electricity of 100,000 diameter 500nm to 2ml centrifuge tube
In, it is mixed with 1ml heparin solution (1mg/ml), acts on 5min.
2, magnetic suck collects nanosphere, and 1ml pure water is washed three times, and magnetic is attached to centrifuge tube bottom, discards liquid.
3,1mlPDADMAC aqueous solution (0.5mg/ml) is added into centrifuge tube, acts on 30min.1ml pure water is washed three times, magnetic
It is adsorbed onto centrifuge tube bottom, discards liquid.
4,1ml heparin solution (1mg/ml) mixing is added into centrifuge tube, acts on 30min.1ml pure water is washed three times, magnetic
It is adsorbed onto centrifuge tube bottom, discards liquid.It is spare.
5,1ml saliva is taken, 2600g is centrifuged 15min, removes cell and cell fragment in body fluid.
6, the saliva after centrifugation is added in the modification nanosphere that step 4 precipitates, room temperature acts on 60min.
7, nanosphere is sunken to centrifuge tube bottom by magnetic suck, discards liquid.
8,1ml 2M NaCl solution is added, after acting on 15min, nanosphere is sunken to centrifuge tube bottom by magnetic suck, and suction contains
The liquid of extracellular vesica, TEM characterization as shown in Figure 10, are consistent with document, and -20 DEG C of preservations are spare.
Claims (6)
1. a kind of method for separating extracellular vesica based on LBL method modification heparin, which is characterized in that specific step is as follows:
(1) method modified layer by layer by LBL first, with the principle of Electrostatic Absorption, on several positively charged nanospheres successively
Alternately modification heparin and diallyl dimethyl ammoniumchloride PDADMAC；Decorative layer is from inside to outside according to heparin, PDADMAC, liver
The sequence of element is arranged, and the number of plies of decorative layer is the odd-level more than or equal to three layers；
(2) by the nanosphere after modification be added in the body fluid containing extracellular vesica react 5min-120min after, centrifugation or
Magnetic suck collects nanosphere；
(3) 2.0~2.5mol/L sodium chloride solution is added in the nanosphere of collection and reacts 10~20min, be adsorbed on nanosphere
The extracellular vesica on surface is eluted in sodium chloride solution, discards nanosphere by centrifugation or magnetic suck, is realized thin in body fluid
The separation of extracellular vesica.
2. the method according to claim 1, wherein positively charged nanosphere is by polyphenyl second in step (1)
Alkene, ferroso-ferric oxide, silicon or silica material nanosphere obtained by amination or silanization treatment.
3. the method according to claim 1, wherein the number of positively charged nanosphere is 50,000-in step (1)
1000000, partial size is between 50 nanometers~10 microns.
4. molten using the aqueous solution or PBS of heparin the method according to claim 1, wherein in step (1)
Liquid modifies heparin on positively charged nanosphere, and the modification time is 1~60min；It is molten using the aqueous solution or PBS of PDADMAC
Liquid modifies PDADMAC on positively charged nanosphere, and the modification time is 1~60min.
5. according to the method described in claim 4, it is characterized in that, in step (1), the aqueous solution of heparin or PBS solution
Concentration is 0.5mg/mL-20mg/mL, and the aqueous solution of PDADMAC or the concentration of PBS solution are 0.5mg/mL-20mg/mL.
6. the method according to claim 1, wherein body fluid is selected from blood plasma, urine, milk, gallbladder in step (2)
It is any in juice or saliva.
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| WO2020199633A1 (en) * | 2018-04-03 | 2020-10-08 | Nanjing Bell Mountain Molecular Medicine Technology Institute Co., Ltd. | Methods for lipid affinity-based non-antibody capture and purification of extracellular vesicles |
| CN112048462A (en) * | 2019-06-05 | 2020-12-08 | 北京丰特云基科技发展有限公司 | Extracellular vesicle separation and enrichment method based on anionic polymer modified matrix |
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| WO2015048566A1 (en) * | 2013-09-26 | 2015-04-02 | The General Hospital Corporation | Methods of isolating extracellular vesicles |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020199633A1 (en) * | 2018-04-03 | 2020-10-08 | Nanjing Bell Mountain Molecular Medicine Technology Institute Co., Ltd. | Methods for lipid affinity-based non-antibody capture and purification of extracellular vesicles |
| CN112048462A (en) * | 2019-06-05 | 2020-12-08 | 北京丰特云基科技发展有限公司 | Extracellular vesicle separation and enrichment method based on anionic polymer modified matrix |
| CN112048462B (en) * | 2019-06-05 | 2022-08-23 | 北京丰特云基科技发展有限公司 | Extracellular vesicle separation and enrichment method based on anionic polymer modified matrix |
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