CN205898536U - Separation and extraction secretes stromatolite centrifugal filtration device of body outward - Google Patents
Separation and extraction secretes stromatolite centrifugal filtration device of body outward Download PDFInfo
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- CN205898536U CN205898536U CN201620793366.3U CN201620793366U CN205898536U CN 205898536 U CN205898536 U CN 205898536U CN 201620793366 U CN201620793366 U CN 201620793366U CN 205898536 U CN205898536 U CN 205898536U
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Abstract
The utility model discloses a separation and extraction secretes stromatolite centrifugal filtration device of body outward for molecular biology and clinical examination technical field, including the filter tube, ultrafiltration pipe and waste liquid collecting pipe, the bottom of filter tube is equipped with the filter membrane, the inside level filtering lumen that forms of filter tube, the bottom of ultrafiltration pipe is equipped with the milipore filter, the ultrafiltration intraduct forms the second grade in the below of filter tube and filters the lumen, it collects the lumen to form the waste liquid in the below of ultrafiltration pipe in the waste liquid collecting pipe, the top mouth of pipe of filter tube is equipped with the one -level and holds in the palm the edge, the top mouth of pipe of ultrafiltration pipe is equipped with the second grade and holds in the palm the edge, the one -level is held in the palm and is followed and second grade support along the last air vent that all is equipped with, the top mouth of pipe of waste liquid collecting pipe is equipped with the block, fashionable can the support one -level of block lid is followed along compressing tightly to hold in the palm in the second grade, hold in the palm the second grade along compressing tightly in the top of the waste liquid collecting pipe mouth of pipe, and through air vent and external intercommunication. The utility model discloses low price, simple operation, the time is short and can carry out the operation of a large amount of samples by the parallel, can satisfy the demand of clinical extensive popularization and application.
Description
Technical field
This utility model is used for molecular biology and clinical testing techniques field, more particularly to a kind of separation and Extraction excretion
The lamination centrifugal filter device of body.
Background technology
Excretion body (exosomes) be a kind of endosome of many bubbles by eukaryotic cell (multivesicular bodies,
Mvbs) it is discharged into the film vesicles of nanometer scale in extracellular environment with after cell membrane fusion, have lipid bilayer structure, greatly
Little about 30 150 nm, are distributed widely in body fluid；Can be secreted by polytype cell, it is contained within different types of albumen
Matter, lipid, mrnas, micrornas and signaling molecule etc. have the material of biologic activity；Has stronger targeting, easy and target cell
Cell membrane merge, biological active agents are optionally delivered to target cell, different iuntercellulars enter row information pass
Pass, adjust intercellular signal transduction, play various biological function.
The extracting method of current excretion body mainly can be summarized as following several: supercentrifugation, the polymer sedimentation method, magnetic bead
Method and ultrafiltration.
First, supercentrifugation is excretion body extracting method most widely used at present, in document concrete centrifugal speed have a little
Difference, generally includes 4 steps: (1) low-speed centrifugal (about 300 × g) removes residual cell；(2) high speed centrifugation (about 16,
500 × g) remove cell debriss；(3) 0.22 μm of membrane filtrations remove major diameter vesicle；(4) ultracentrifugation (about 120,000
× g) precipitation excretion body, can be selected for sucrose density gradient centrifugation for improving purity.
Ultracentrifugation extraction method mainly has the disadvantage that:
1) time-consuming for operation: takes about 8-24 h from processing the biological fluid completing to collecting excretion body overall process；
2) low separation efficiency: educational circles is proved the response rate only about 10% to excretion body for the method at present, obtain enough
The sample of analysis needs larger samples amount；
3) need particular instrument: extract supercentrifuge used and be not belonging to Routine Test Lab configuration equipment；
4) parallel processing sample number is limited: according to different centrifuge rotors, is capable of the sample size one of operation repetitive simultaneously
As be not more than 6.
2nd, the polymer sedimentation method are a kind of excretion body extracting method of newer type, due to which overcoming supercentrifugation
Many defects, are widely used in scientific research field at present, and existing dependent merchandise reagent is sold.Its realization is mainly passed through will
Sample is mixed according to a certain percentage with polymer stock solution, and low speed (about 10,000 × g) is used in incubation a period of time (about 30 min) afterwards
Precipitate is collected by centrifugation, after adding isotonic buffer solution dissolution precipitation, is excretion body.
The major defect of the polymer sedimentation method is as follows:
1) experimental cost is high: with main flow commercial reagents, the such as product of system biosciences (sbi) company
As a example exoquick-tc, the reagent consumption extracted every time, more than 180 yuans, is unfavorable for that large-scale promotion makees clinical practice；
2) limited by subsequent treatment: because extraction process needs to add in the sample added ingredient, after the excretion body collected
Continue and can not be directly used as the purposes such as cell culture or feedback；
3) it is not suitable for bulk sample: because its principle and higher cost lead to the method to be not particularly suited for large volume
The process of sample.
3rd, the excretion body extracting method of another kind of main flow is paramagnetic particle method.The method relies on the magnetic bead being fixed with specific antibodies
With sample incubation, combined with the excretion body having corresponding film surface antigen in sample using antigen antibody reaction, finally utilize magnetic force
Magnetic bead is together adsorbed with excretion body, realizes the extraction of excretion body.
Paramagnetic particle method equally has the obvious disadvantage that:
1) experimental cost is high: magnetic bead needs special working ability, extracts the crosslinking of required antibody and antibody and magnetic bead simultaneously
The cost of implementation of the method is further increased；
2) it is only capable of extracting a certain specific excretion body subgroup in sample: because the method is based on antigen antibody reaction, therefore only
Specific extraction can go out to have the excretion body subgroup of a certain known film surface antigen from sample；
3) it is not suitable for bulk sample: because its principle and higher cost lead to the method to be not particularly suited for large volume
The process of sample.
4th, also once carried out excretion body enrichment using ultrafiltration during part is studied, but the method for its report needed ultracentrifugation
Precipitate excretion body further, operating process is tediously long, is not appropriate for Clinical practice.And there is no the ultrafiltration that can apply to serum/plasma
The method extracting excretion body.
In sum, there is no cheap, simple operation at present, the time is short and parallel can carry out the outer of great amount of samples operation
Secrete body extracting method and device disclosure satisfy that the demand of clinical large-scale promotion application.
Utility model content
For solving the above problems, this utility model provides a kind of lamination centrifugal filter device of separation and Extraction excretion body, valency
Lattice are cheap, simple operation, and the time is short and parallel can carry out great amount of samples operation.
This utility model solve its technical problem be the technical scheme is that a kind of lamination of separation and Extraction excretion body from
Heart defecator, including filter tube, is enclosed within the super filter tube outside filter tube and is enclosed within waste collection pipe outside super filter tube, described
The bottom of filter tube is provided with filter membrane, and filter tube is internally formed single filter tube chamber, and the bottom of super filter tube is provided with ultrafilter membrane, super filter tube
Inside is in the cascade filtration tube chamber formed below of filter tube, waste collection pipe in the waste collection pipe formed below of super filter tube
Chamber, the top mouth of pipe of described filter tube is provided with one-level support edge, and the top mouth of pipe of super filter tube is provided with two grades of support edges, one-level support edge and two
Level support is provided with block along being above equipped with airway, the top mouth of pipe of waste collection pipe, can be by one-level support along compression when block covers
In two grades of support edges, two grades are held in the palm along the top mouth of pipe being pressed in waste collection pipe, single filter tube chamber and cascade filtration tube chamber are equal
It is in communication with the outside by airway.
It is further used as the improvement of technical solutions of the utility model, the aperture of described filter membrane is not more than 1 μm.
It is further used as the improvement of technical solutions of the utility model, ultrafilter membrane is 30-100 kda granule for molecular cut off
Ultrafilter membrane.
It is further used as the improvement of technical solutions of the utility model, left side tube wall below filter tube for the super filter tube and right side
Tube wall is the plane tube wall tilting to middle part, and between two plane tube walls, Formation cross-section is in the cascade filtration tube chamber of v shape, and at least one is flat
Ultrafilter membrane window is opened up on facial canal wall, ultrafilter membrane window embeds hyperfiltration membrane assembly, described hyperfiltration membrane assembly includes ultrafilter membrane to be fixed
Plate and the ultrafilter membrane being attached inside ultrafilter membrane fixed plate, the lower end of described ultrafilter membrane fixed plate is provided with some tap holes.
It is further used as the improvement of technical solutions of the utility model, block adopts screw thread with the top mouth of pipe of waste collection pipe
Connect or snap connection.
The beneficial effects of the utility model: when this utility model uses, add the incubation of 0.5 times of volume to delay in the sample
Rush liquid, and add 0.05 times of volume of protein enzyme k liquid storage, fully mix, incubated at room 8-15 min；The sample having processed is shifted
To filter tube of the present utility model, centrifugation, mix, collect the trapped fluid in super filter tube, that is, complete the separation and Extraction of excretion body.
This utility model is cheap, simple operation, and the time is short and parallel can carry out great amount of samples operation, disclosure satisfy that clinical extensive
The demand of popularization and application.
Brief description
The utility model is described in further detail below in conjunction with the accompanying drawings:
Fig. 1 is this utility model structural representation.
Specific embodiment
With reference to Fig. 1, that show the concrete structure of the preferred embodiment of this utility model.Will be detailed below this reality
With the construction featuress of new each element, and during if there are being described to direction (upper and lower, left and right, before and after), it is with shown in Fig. 1
Structure be that but actually used direction of the present utility model is not limited thereto with reference to description.
This utility model provides a kind of lamination centrifugal filter device of separation and Extraction excretion body, including filter tube 1, is enclosed within
The super filter tube 2 in filter tube 1 outside and the waste collection pipe 3 being enclosed within super filter tube 2 outside, the bottom of described filter tube 1 is provided with filter membrane
11, filter tube 1 is internally formed single filter tube chamber 12, and the bottom of super filter tube 2 is provided with ultrafilter membrane 21, in filtration inside super filter tube 2
The cascade filtration tube chamber 22 formed below of pipe 1, in the waste collection tube chamber 31 formed below of super filter tube 2, institute in waste collection pipe 3
The top mouth of pipe stating filter tube 1 is provided with one-level support along 13, and the top mouth of pipe of super filter tube 2 is provided with two grades of supports along 23, and one-level support is along 13
It is equipped with airway with two grades of supports along along 23, waste collection pipe 3 is matching used centrifuge tube with centrifugation apparatus, waste collection pipe
The 3 top mouth of pipe is provided with block, and the top mouth of pipe of block and waste collection pipe 3 is using threadeding or snap connection.Work as block
One-level support can be pressed in two grades of supports along 23, by two grades of supports along the 23 top mouths of pipe being pressed in waste collection pipe 3 along 13 when covering,
Meanwhile, single filter tube chamber 12 and cascade filtration tube chamber 22 are all in communication with the outside by airway, it is to avoid air pressure problem leads to difficulty
To filter.
Super filter tube 2 is, in the left side tube wall of filter tube 1 lower section and right side tube wall, the plane tube wall tilting to middle part, and two is flat
Between facial canal wall, Formation cross-section is in the cascade filtration tube chamber 22 of v shape, and the outer wall of cascade filtration tube chamber 22 is provided with scale.At least one is flat
Ultrafilter membrane window is opened up on facial canal wall, ultrafilter membrane window embeds hyperfiltration membrane assembly, described hyperfiltration membrane assembly includes ultrafilter membrane to be fixed
Plate and the ultrafilter membrane 21 being attached inside ultrafilter membrane fixed plate, the lower end of described ultrafilter membrane fixed plate is provided with some tap holes.
Method using this utility model separation and Extraction excretion body is as follows:
1) add the incubation buffer of 0.5 times of volume in the sample, and add 0.05 times of volume of protein enzyme k liquid storage, fully
Mix, incubated at room 8-15 min；
2) sample that step 1) has been processed is transferred in the filter tube 1 of lamination centrifugal filter device, centrifugation, mixes, receive
Trapped fluid in collection super filter tube 2.
When test serum sample is added in filter tube, under the action of the centrifugal, serum sample passes through the filter of filter tube 1 bottom
Film 11, conventional filter membrane 11 is microporous filter membrane, and aperture is 0.22 μm.Most microcapsule bubbles are trapped within filter tube 1.And
Excretion body, soluble protein, high-abundance proteins enzymatic fragment etc. have then passed through this metafiltration film and have entered in super filter tube 2.Due to super
The lower section of chimney filter 2 tube wall is ultrafilter membrane 21, and bottom tool cascade filtration tube chamber 22, in centrifugal process, solubility portion in serum
Divide, high-abundance proteins enzymatic fragment and more small granule ooze out from the ultrafilter membrane 21 of side wall, enter waste collection pipe 3.Due to
The bottom tool cascade filtration tube chamber 22 of super filter tube 2, by member-retaining portion liquid in centrifugal process, excretion body can be protected in this solution
Deposit.After being centrifuged, take out super filter tube 2, make liquid blending in reservation chamber with pipettor piping and druming, collect trapped fluid and be outside end-product
Secrete liquid solution.
Preferably, if desired improve excretion body purity, dbps can be added toward in super filter tube 2,3,000 × g is centrifuged 10 min,
For removing some soluble proteins, abandon filter liquor and be repeated once.
Preferably, mix and collect trapped fluid as excretion liquid solution, subsequent treatment can be carried out.
Wherein, the aperture of described filter membrane 11 is not more than 1 μm, is preferably 0.22 μm in the present embodiment.Ultrafilter membrane 21 is retention
Molecular weight is the ultrafilter membrane of 30-100 kda granule.Conventional filter sizes have 0.1-100 μm.In serum, common chaff interference is
High-abundance proteins and microcapsule bubble (microvesicles, mvs), through pretreatment, a large amount of high-abundance proteins can be by protease k enzyme
Solution becomes small fragment.And microcapsule bulb diameter is about 200-1000 nm, can remove the microcapsule of the overwhelming majority with 0.22 μm of filter membrane herein
The interference of bubble.Microcapsule bubble is entered the major impurity in cascade filtration chamber by membrane retention in the single filter chamber in filter tube
Predominantly the high-abundance proteins in serum are by the polypeptide fragment of protease k enzyme action.It is about 5- because polypeptide fragment mostly is molecular weight
The linear molecule of 10 kda, therefore the ultrafilter membrane from 30 more than kda can be filtered.Meanwhile, the ultrafiltration fenestra in the range of this
Footpath is significantly smaller than excretion body diameter, and excretion body will be retained in single filter intracavity.
Method is compared with supercentrifugation:
A) this method is obtained in that the excretion body of purity suitable with supercentrifugation；
B) this method all operationss can complete in 30 min, greatly shortens the operating time；
C) all processes can ambient operation, easy and simple to handle；
D) this method can complete in any laboratory equipped with desktop compact centrifuge, to other experimental facilitiess substantially no
Demand；
E) according to centrifuge model, the present invention can process multiple samples simultaneously, and laboratory common small table centrifuge is joined
Standby rotor just can complete the extraction of up to 24 samples simultaneously.
Compared with the polymer sedimentation method:
A) this method is obtained in that the excretion body of purity suitable with the polymer sedimentation method；
B) this method consumables cost is more cheap；
C) the excretion body product obtaining can be the purposes such as cell culture or feedback substantially without processing further.
Compared with paramagnetic particle method:
A) this utility model consumables cost is more cheap；
B) this utility model can extract whole subgroups of excretion body in sample, does not rely on specific film surface antigen.
Certainly, the invention is not limited to above-mentioned embodiment, and those of ordinary skill in the art are without prejudice to this
Equivalent variations or replacement also can be made, these equivalent modifications or replacement are all contained in the application on the premise of utility model spirit
In claim limited range.
Claims (5)
1. a kind of lamination centrifugal filter device of separation and Extraction excretion body it is characterised in that: include filter tube, be enclosed within outside filter tube
The super filter tube of side and the waste collection pipe being enclosed within outside super filter tube, the bottom of described filter tube is provided with filter membrane, the internal shape of filter tube
Become single filter tube chamber, the bottom of super filter tube is provided with ultrafilter membrane, in the cascade filtration pipe formed below of filter tube inside super filter tube
Chamber, in the waste collection tube chamber formed below of super filter tube in waste collection pipe, the top mouth of pipe of described filter tube is provided with one-level support
Edge, the top mouth of pipe of super filter tube is provided with two grades of support edges, and one-level support edge and two grades of support edges are above equipped with airway, waste collection pipe
The top mouth of pipe is provided with block, when block covers can by one-level support along be pressed in two grades support along, by two grades support along be pressed in waste liquid receive
The top mouth of pipe of collector, single filter tube chamber and cascade filtration tube chamber are all in communication with the outside by airway.
2. separation and Extraction excretion body according to claim 1 lamination centrifugal filter device it is characterised in that: described filter membrane
Aperture be not more than 1 μm.
3. separation and Extraction excretion body according to claim 1 lamination centrifugal filter device it is characterised in that: ultrafilter membrane is
Molecular cut off is the ultrafilter membrane of 30-100 kda granule.
4. separation and Extraction excretion body according to claim 1 lamination centrifugal filter device it is characterised in that: super filter tube in
Left side tube wall below filter tube and right side tube wall are the plane tube wall tilting to middle part, and between two plane tube walls, Formation cross-section is in
The cascade filtration tube chamber of v shape, at least one plane tube wall opens up ultrafilter membrane window, and ultrafilter membrane window embeds hyperfiltration membrane assembly, institute
State the ultrafilter membrane that hyperfiltration membrane assembly includes ultrafilter membrane fixed plate and is attached inside ultrafilter membrane fixed plate, described ultrafilter membrane fixed plate
Lower end be provided with some tap holes.
5. separation and Extraction excretion body according to claim 1 lamination centrifugal filter device it is characterised in that: block with useless
The top mouth of pipe of liquid collecting pipe is using threaded or snap connection.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620793366.3U CN205898536U (en) | 2016-07-26 | 2016-07-26 | Separation and extraction secretes stromatolite centrifugal filtration device of body outward |
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| CN201620793366.3U CN205898536U (en) | 2016-07-26 | 2016-07-26 | Separation and extraction secretes stromatolite centrifugal filtration device of body outward |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106124282A (en) * | 2016-07-26 | 2016-11-16 | 广州海力特生物科技有限公司 | A kind of method secreting body outside lamination centrifugal filtration separation and Extraction |
| EP3444029A1 (en) * | 2017-08-17 | 2019-02-20 | Sartorius Stedim Biotech GmbH | Protein purification device |
| CN111426542A (en) * | 2020-05-12 | 2020-07-17 | 山东省海洋资源与环境研究院 | A separation device suitable for different groups of plankton |
| CN111879595A (en) * | 2020-08-31 | 2020-11-03 | 山西医科大学第二医院 | A red blood cell cleaning device |
-
2016
- 2016-07-26 CN CN201620793366.3U patent/CN205898536U/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106124282A (en) * | 2016-07-26 | 2016-11-16 | 广州海力特生物科技有限公司 | A kind of method secreting body outside lamination centrifugal filtration separation and Extraction |
| WO2018018707A1 (en) * | 2016-07-26 | 2018-02-01 | 广州海力特生物科技有限公司 | Multi-layered centrifugal filter for exosome extraction |
| CN106124282B (en) * | 2016-07-26 | 2019-07-16 | 广州海力特生物科技有限公司 | A kind of method of lamination centrifugal filtration separation and Extraction excretion body |
| US11684893B2 (en) | 2016-07-26 | 2023-06-27 | Guangzhou Supbio Bio-Technology And Science Co., Ltd. | Method for exosome separation and extraction by stacked centrifugal filtration |
| EP3444029A1 (en) * | 2017-08-17 | 2019-02-20 | Sartorius Stedim Biotech GmbH | Protein purification device |
| CN111426542A (en) * | 2020-05-12 | 2020-07-17 | 山东省海洋资源与环境研究院 | A separation device suitable for different groups of plankton |
| CN111879595A (en) * | 2020-08-31 | 2020-11-03 | 山西医科大学第二医院 | A red blood cell cleaning device |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200508 Address after: Room 301, 3 / F, building 5, Dongguan Taiwan Biotechnology cooperation center, Songshanhu hi tech Industrial Development Zone, Dongguan, Guangdong 523808 Patentee after: Dongguan Microprecision Testing Research Institute Co.,Ltd. Address before: 510535, A7 building, No. 11, Kaiyuan Avenue, Guangzhou hi tech Industrial Development Zone, Guangdong, two Patentee before: Guangzhou Supbio Bio-technology and Science Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| CP03 | Change of name, title or address |
Address after: Room 301, Building 5, No. 1 Taoyuan Road, Songshanhu Park, Dongguan City, Guangdong Province, 523808 Patentee after: Dongguan Microprecision Testing Research Institute Co.,Ltd. Country or region after: China Address before: 523808 Room 301, 3 / F, building 5, Dongguan Taiwan Biotechnology cooperation and breeding center, Songshanhu high tech Industrial Development Zone, Dongguan City, Guangdong Province Patentee before: Dongguan Microprecision Testing Research Institute Co.,Ltd. Country or region before: China |
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| CP03 | Change of name, title or address |