CN107597216A - A kind of method that excretion body is separated using micro-fluid chip - Google Patents
A kind of method that excretion body is separated using micro-fluid chip Download PDFInfo
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- CN107597216A CN107597216A CN201710158638.1A CN201710158638A CN107597216A CN 107597216 A CN107597216 A CN 107597216A CN 201710158638 A CN201710158638 A CN 201710158638A CN 107597216 A CN107597216 A CN 107597216A
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Abstract
A kind of method that excretion body is separated using micro-fluid chip, the present invention relates to a kind of method that excretion body is captured using microfluid biochip.This method comprises the following steps：Processing early stage of sample, including the concentration of sample is adjusted and preliminary purification.The sample of preliminary treatment is passed through excretion body capture chip and further separates excretion body.The micro-fluidic chip combines microarray post and the affine identification biomolecule of specificity, realizes the capture to excretion body in cells and supernatant and Human Fluids.The excretion body of separation can be further used for scientific research and clinical analysis.
Description
Technical field
The present invention relates to biological technical field, is specifically designed the method using micro-fluid chip separation excretion body.
Background technology
Excretion body (exosomes) is that a kind of diameter is about the vesica that 30-150nm has double-deck plasma structure, is led to by cell
Exocytosis is crossed to discharge into extracellular gap or biological body fluid.In recent years, excretion body receives more and more attention, and turns into new
Study hotspot, includes the various biomolecules such as mRNAs, microRNAs and protein specific to derived cell in excretion body,
Played an important role in signal transduction and immune system, more there are some researches show the transfer of excretion body and tumour, the nervous system disease
Etc. closely related, its content is also the marker molecule of a variety of Disease Clinicals detections and diagnosis.
The isolation technics of excretion body includes supercentrifugation, filter centrifugation method, Density ultracentrifugation, immune at present
Magnetic bead combination supercentrifugation and chromatography etc..These technologies require instrument and equipment high, cumbersome time-consuming, it is therefore desirable to have
The excretion body capture technique of effect solves these problems.
Based on described above, the present invention intend application modified biologic specificity molecule the capture of microarray post fluid chip it is outer
Body is secreted, can be facilitated, timesaving separation excretion body, applied to scientific research and clinic.
The content of the invention
Object of the present invention is to provide a kind of method that excretion body is separated using micro-fluid chip, the micro-fluidic chip
Microarray post and the affine identification biomolecule of specificity are combined, is realized to excretion body in cells and supernatant and Human Fluids
Capture.
Technical scheme：A kind of method that excretion body is separated using micro-fluid chip, comprises the following steps：
1), the pre-treatment of sample：Cells and supernatant or human body fluid sample are collected under aseptic condition, sample is adjusted with PBS
This concentration, sample is carried out by centrifugally operated tentatively to go the removal of impurity；0.22um filtering with microporous membrane samples are used again, further
Go the removal of impurity；
2) excretion body, is separated：Sample Jing Guo preliminary treatment is passed through micro-fluid chip by injector or sampling pump, entered
Row specific isolation captures excretion body.
Used micro-fluid chip is with snakelike split tunnel, and cross arrangement is set on the snakelike split tunnel
Micro-pillar array, can be one kind in the polygons such as circle, triangle, quadrangle at the top of the micro-pillar array.
300 × g is abandoned after centrifuging 10min, then 2000 × g centrifugations 20min under the conditions of the pre-treating method of the sample is 4 degree
Precipitation, l0,000 × g abandon precipitation after centrifuging 30min；Again with 0.22 μm of membrane filtration.
The concentration of PBS used is 0.01mol/L.
The flow velocity that the sample is passed through chip is 5-50ul/min.
Authentication method used is that ESEM and Western Blot are analyzed.
The advantage of the invention is that：It is simple to operate using the micro-fluid chip of modified specificity biomolecule, reduce
The consumption of sample and reagent, separation purity is high, reduces cost.
Brief description of the drawings
Fig. 1 separates excretion swept-volume Electronic Speculum qualification figure；
Fig. 2 separates excretion body western Blot qualification figures；
Fig. 3 separates excretion swept-volume Electronic Speculum qualification figure；
Fig. 4 separates excretion body western Blot qualification figures；
Fig. 5 separates excretion swept-volume Electronic Speculum qualification figure；
Fig. 6 separates excretion body western Blot qualification figures.
Embodiment
Below with regard to embodiment, the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate this hair
Bright rather than limitation the scope of the present invention, after the present invention has been read, those skilled in the art are to of the invention various etc.
The modification of valency form falls within the application appended claims limited range.In order to understand the present invention, below to the present invention
It is described in further detail.
A kind of method that excretion body is separated using micro-fluid chip, comprises the following steps：
1), the pre-treatment of sample：Cells and supernatant or human body fluid sample are collected under aseptic condition, sample is adjusted with PBS
This concentration, sample is carried out by centrifugally operated tentatively to go the removal of impurity；0.22um filtering with microporous membrane samples are used again, further
Go the removal of impurity；
2) excretion body, is separated：Sample Jing Guo preliminary treatment is passed through micro-fluid chip by injector or sampling pump, entered
Row specific isolation captures excretion body.
Used micro-fluid chip is with snakelike split tunnel, and cross arrangement is set on the snakelike split tunnel
Micro-pillar array, can be one kind in the polygons such as circle, triangle, quadrangle at the top of the micro-pillar array.
300 × g is abandoned after centrifuging 10min, then 2000 × g centrifugations 20min under the conditions of the pre-treating method of the sample is 4 degree
Precipitation, l0,000 × g abandon precipitation after centrifuging 30min；Again with 0.22 μm of membrane filtration.
The concentration of PBS used is 0.01mol/L.
The flow velocity that the sample is passed through chip is 5-50ul/min.
Authentication method used is that ESEM and Western Blot are analyzed.
Embodiment 1
The embodiment is to carry out excretion body separation to HepG2 cell culture mediums supernatant using micro-fluid chip；Chip
Sample holes and go out sample sky and link conduit, sample holes conduit is linked by injector and sampling pump；By HepG2 cells and supernatants
In 4 degree of centrifugations, 300 × g 10min, 2000 × g 20min, precipitation is abandoned, remove cell；Then l0,000 × g 30min, it is heavy to abandon
Form sediment, remove subcellular component；Learnt from else's experience the cell culture fluid of pre-treatment, and be passed through in chip, adjust flow velocity；After the completion of separation, use
The 0.01mol/l PBS of sterilizing rinse chip.
The morphosis of the excretion body of electron microscopic observation chip capture；Taking sample to be resuspended in 100ulPBS (takes 10ul to dilute
Into 30ul, then 10ul is taken to be diluted to 30ul)；Absorption 10ul sample drops, which are added on 2mm load sample copper mesh, precipitates 1min, and filter paper is drawn floating
Liquid, draw 3% sodium phosphotungstate solution (PH6.8) 10ul and be added dropwise on copper mesh, room temperature negative staining 5min, filter paper draws supernatant liquid, double steamings
Washing 1-2 dries all over rear normal temperature；Upper machine, in electric Microscopic observation and take a picture, 80kv-120kv imagings.
Excretion body protein extract solution is passed through chip, fully after reaction, collects lysate.
Appropriate protein extract is taken, western analyses are carried out from tsg100 primary antibodies.
Embodiment 2
The embodiment is to carry out excretion body separation to MCF-7 cell culture mediums supernatant using micro-fluid chip；Chip
Sample holes and go out sample sky and link conduit, sample holes conduit is linked by injector and sampling pump；By MCF-7 cells and supernatants
In 4 degree of centrifugations, 300 × g 10min, 2000 × g 20min, precipitation is abandoned, remove cell；Then l0,000 × g 30min, it is heavy to abandon
Form sediment, remove subcellular component；Learnt from else's experience the cell culture fluid of pre-treatment, and be passed through in chip, adjust flow velocity；After the completion of separation, use
The 0.01mol/l PBS of sterilizing rinse chip.
The morphosis of the excretion body of electron microscopic observation chip capture；Taking sample to be resuspended in 100ulPBS (takes 10ul to dilute
Into 30ul, then 10ul is taken to be diluted to 30ul), absorption 10ul sample drops, which are added on 2mm load sample copper mesh, precipitates 1min, and filter paper is drawn floating
Liquid, draw 3% sodium phosphotungstate solution (PH6.8) 10ul and be added dropwise on copper mesh, room temperature negative staining 5min, filter paper draws supernatant liquid, double steamings
Washing 1-2 dries all over rear normal temperature；Upper machine, in electric Microscopic observation and take a picture, 80kv-120kv imagings.
Excretion body protein extract solution is passed through chip, fully after reaction, collects lysate.
Appropriate protein extract is taken, western analyses are carried out from tsg100 primary antibodies.
Embodiment 3
The embodiment is to carry out excretion body separation to human plasma using micro-fluid chip；Chip sample holes and to go out sample empty
Conduit is linked, sample holes conduit is linked by injector and sampling pump；By blood plasma in 4 degree of centrifugations, 300 × g 10min, 2000 ×
G 20min, collect supernatant；Then l0,000 × g 30min, precipitation is abandoned, removes subcellular component；Learnt from else's experience the blood of pre-treatment
Slurry, is passed through in chip, adjusts flow velocity；After the completion of separation, chip is rinsed with the 0.01mol/l PBS of sterilizing.
The morphosis of the excretion body of electron microscopic observation chip capture；Taking sample to be resuspended in 100ulPBS (takes 10ul to dilute
Into 30ul, then 10ul is taken to be diluted to 30ul), absorption 10ul sample drops, which are added on 2mm load sample copper mesh, precipitates 1min, and filter paper is drawn floating
Liquid, draw 3% sodium phosphotungstate solution (PH6.8) 10ul and be added dropwise on copper mesh, room temperature negative staining 5min, filter paper draws supernatant liquid, double steamings
Washing 1-2 dries all over rear normal temperature；Upper machine, in electric Microscopic observation and take a picture, 80kv-120kv imagings.
Excretion body protein extract solution is passed through chip, fully after reaction, collects lysate.
Appropriate protein extract is taken, western analyses are carried out from tsg100 primary antibodies.
In the present invention use modified specificity biomolecule micro-fluid chip, it is simple to operate, reduce sample and
The consumption of reagent, separation purity is high, reduces cost.
Claims (6)
- A kind of 1. method that excretion body is separated using micro-fluid chip, it is characterised in that comprise the following steps：1), sample preprocessing：Cells and supernatant or human body fluid sample are collected under aseptic condition, the dense of sample is adjusted with PBS Degree, sample is carried out by centrifugally operated tentatively to go the removal of impurity；Sample is further gone with 0.22um miillpore filters again The removal of impurity；2) excretion body, is separated：Sample Jing Guo preliminary treatment is passed through micro-fluid chip by injector or sampling pump, carried out special Different in nature separating trap excretion body.
- 2. a kind of method that excretion body is separated using micro-fluid chip according to claim 1, it is characterised in that used Micro-fluid chip be the micro-pillar array that cross arrangement is set on the snakelike split tunnel with snakelike split tunnel, institute It is one kind in the polygons such as circle, triangle, quadrangle to state at the top of micro-pillar array.
- A kind of 3. method that excretion body is separated using micro-fluid chip according to claim 1, it is characterised in thatThe step of sample preprocessing method：300 × g centrifuges 10min, rear 2000 × g centrifugations under the conditions of 4 degree of centrifuge Abandon precipitation after 20min, then precipitation is abandoned after l0000 × g centrifugations 30min, then with 0.22 μm of membrane filtration.
- 4. a kind of method that excretion body is separated using micro-fluid chip according to claim 1, it is characterised in that used The concentration of PBS is 0.01mol/L.
- A kind of 5. method that excretion body is separated using micro-fluid chip according to claim 1, it is characterised in that the sample Originally the flow velocity for being passed through chip is 5-50ul/min.
- A kind of 6. method that excretion body is separated using micro-fluid chip according to claim 1, it is characterised in that mirror used Determine method to analyze for ESEM and Western Blot.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872564A (en) * | 2018-09-12 | 2018-11-23 | 杭州多泰科技有限公司 | A kind of external instant detection platform and its detection method based on excretion body |
| CN110320355A (en) * | 2018-03-30 | 2019-10-11 | 上海市肿瘤研究所 | A kind of micro-fluidic chip and the detection method for recycling excretion body |
| CN110339874A (en) * | 2019-06-21 | 2019-10-18 | 中国科学院上海微系统与信息技术研究所 | A kind of separation of excretion body and surface protein detection micro fluidic device and application method |
| CN113462520A (en) * | 2021-07-26 | 2021-10-01 | 百奥芯(苏州)生物科技有限公司 | Circulating tumor exosome enrichment chip and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110320355A (en) * | 2018-03-30 | 2019-10-11 | 上海市肿瘤研究所 | A kind of micro-fluidic chip and the detection method for recycling excretion body |
| CN108872564A (en) * | 2018-09-12 | 2018-11-23 | 杭州多泰科技有限公司 | A kind of external instant detection platform and its detection method based on excretion body |
| CN110339874A (en) * | 2019-06-21 | 2019-10-18 | 中国科学院上海微系统与信息技术研究所 | A kind of separation of excretion body and surface protein detection micro fluidic device and application method |
| CN113462520A (en) * | 2021-07-26 | 2021-10-01 | 百奥芯(苏州)生物科技有限公司 | Circulating tumor exosome enrichment chip and application thereof |
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Effective date of registration: 20200717 Address after: 225000 Yangzhou high tech Industrial Development Zone, Jiangsu Province West Road, No. 217 Applicant after: JIANGSU YINUOWAN CELL CLINIC Co.,Ltd. Address before: 225127 No. 217 West Development Road, Jiangsu, Yangzhou Applicant before: Li Fangfang |
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