CN107365737A - A kind of method based on silanization lipid probe separation excretion body - Google Patents
A kind of method based on silanization lipid probe separation excretion body Download PDFInfo
- Publication number
- CN107365737A CN107365737A CN201710579129.6A CN201710579129A CN107365737A CN 107365737 A CN107365737 A CN 107365737A CN 201710579129 A CN201710579129 A CN 201710579129A CN 107365737 A CN107365737 A CN 107365737A
- Authority
- CN
- China
- Prior art keywords
- lipid
- excretion body
- peg
- silane
- glassware
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a kind of method based on silanization lipid probe separation excretion body.Silane-PEG- lipid moleculars are first added in glassware by it so that silane-PEG- lipid moleculars pass through silicon oxygen bond and glassware surface silicon covalent bond, PEG- lipid moleculars modification glass surface；Then excretion body is added, PEG- lipid moleculars are inserted into the phospholipid bilayer of excretion body, and then excretion body is fixed on into glassware surface, realize the separation of excretion body.The beneficial effects of the present invention are：By silane by lipid probe molecular modification to glass surface, the adsorbing separation of excretion body is realized, method is simple, can be easily separated.
Description
Technical field
The invention belongs to field of biomedicine technology, is related to a kind of side based on silanization lipid probe separation excretion body
Method.
Background technology
Excretion body is a kind of extracellular vesica of cell active secretion, has phospholipid bilayer Rotating fields, diameter~30-
100nm, density~1.10-1.19g/ml are in the form of annular discs.Research confirms that excretion body can be used as courier, pass through blood, urine, breast
The conduction of signal between the approach mediated cell such as juice, saliva.Although the formation mechenism of excretion body is not still especially clear,
Research finds that excretion body carries various albumen, nucleic acid, or even the DNA of full-length genome.It is thus tolerant in excretion body generally can be anti-
The state of cell health or disease is reflected, and can be to the affecting cells of surrounding.More and more research shows, excretion body
By the influence to tumor microenvironment, the generation, development and transfer of tumour have directly been mediated.Therefore, it is tolerant in excretion body
The relevant information of tumour is reflected to a certain extent, is a kind of potential tumor cells mark for lesion detection.
At present, although having there is the related work that substantial amounts of article reports excretion body, the research for excretion body
Bottleneck all the time be present with application, be mainly manifested in excretion body and separate and collect aspect.The main basis of the isolation technics of excretion body at present
What its own possessed physics and Biological characteristics developed, including：Supercentrifugation based on density, based on size
It is separated by filtration method, the immunity enrichment method based on antibody, based on PEG precipitation method similar to isolated viral etc..Although these methods pair
The separation of excretion body plays a significant role, but the shortcomings that more serious also all be present.It is first for supercentrifugation
First ultracentrifuge and consumptive material are expensive, and only seldom laboratory can be born, even more with high costs into application field.
Secondly separation needs tens of hours, takes longer.And isolated sample also has, and protein contamination is serious, excretion body is broken
The presence for the problems such as splitting.Although the method for being separated by filtration can efficiently separate to obtain the higher excretion body of purity, separation process
Middle protein, excretion body are easily adsorbed in seperation film or splitter, cause to block, and material consumption is larger, are taken also longer.Simultaneously
When excretion body passes through duct, also it is very easy to be squeezed rupture.Immunity enrichment method can be with isolated purity highest
Excretion body, but shortcoming is also apparent.For antibody itself, preparation process is extremely complex, price costly, and
And the problems such as quality is unstable between batch be present.Need to use substantial amounts of antibody when separating excretion body, cost is higher, separative efficiency
Lowly.Presently the most simple and easy method is the PEG precipitation method, can low cost more quickly isolated excretion body, base
In this separation method, You Duo companies develop excretion body separating kit, but protein contamination is still very tight
Weight, significantly impacts follow-up experiment.Although the methods of can passing through ultracentrifugation, purifies again to product, there is still a need for
The shortcomings that considering supercentrifugation.Recently, article reports a kind of method for separating excretion body by lipid probe, principle is
Using the principle of PEG- lipid derivates probe and excretion body film similar compatibility, after lipid probe is inserted into excretion body film,
Using magnetic bead by biotin-avidin specific bond, the magnetic bead with excretion body is separated from sample, then by putting
The effect of changing, excretion body is replaced from magnetic bead, carry out follow-up experiment.Although the method separating rate is very fast, obtain
Sample purity is higher, but is that magnetic bead, biotin and Avidin dosage are larger the problem of exist, and cost is very high, and step compared with
To be cumbersome.
The content of the invention
For overcome the deficiencies in the prior art, it is an object of the invention to provide a kind of cost it is low, it is excellent effect based on
The method that silanization lipid probe separates excretion body.
Technical scheme is specifically described as follows.
A kind of method based on silanization lipid probe separation excretion body, silane-PEG- lipid moleculars are first added to by it
In glassware so that silane-PEG- lipid moleculars pass through silicon oxygen bond and glassware surface silicon covalent bond, PEG- lipids point
Son modification glass surface；Then excretion body is added, PEG- lipid moleculars are inserted into the phospholipid bilayer of excretion body, and then will
Excretion body is fixed on glassware surface, realizes the separation of excretion body；Wherein：The structure of the silane-PEG- lipid moleculars is such as
Shown in following formula：
Wherein：The lipid molecular is monoacyl lipid, any in diacyl lipid or cholesterol, monoacyl lipid or
The carbochain of diacyl lipid is between C10 to C26.
In the present invention, PEG containsIndividual oxirane EO.
The method of the present invention, is comprised the following steps that：
(1) glassware is cleaned using plasma cleaner cleaning 2-10 minutes to surface；
(2) at room temperature, silane-PEG- lipid moleculars are dissolved into absolute ethyl alcohol, and it is 1pmol~1 μm ol's to obtain concentration
Silane-PEG- lipid solns；
(3) silane-PEG- lipid solns are added in glassware, lucifuge, room temperature, are incubated 1~5 minute, discard and repair
Adorn liquid, a large amount of pure water rinsings；
(4) after glassware naturally dry, 55~65 DEG C of baking oven, toast 1~2 minute, it is standby；
(5) fluid sample is added to after the modification that step (4) obtains in glassware, and 37 DEG C are incubated 2 minutes, fluid sample
Including blood plasma, saliva and urine；
(6) solution example is discarded, PBS is rinsed three times；Realize the separation of excretion body.
Compared to the prior art, the beneficial effects of the present invention are：
The present invention realizes lipid probe molecular modification to glass surface by silane the adsorbing separation of excretion body, avoids
The use of the price expensive reagent such as magnetic bead, biotin and Avidin, greatlys save separation costs, and simplifies experiment step
Suddenly.The separation method of the present invention, can be to mark and separate in the short period to excretion body, it is often more important that, this method allows each
The follow-up analysis of molecules of kind, including Elisa, western blot, the extraction of genome, identification, amplification and sequencing.Wherein to glass
The modification of ware disposably can be modified largely, the glass dish after modification can long-time Room-temperature seal preserve, for next time use
The separation time saves time.
Brief description of the drawings
Fig. 1 is the present invention using the schematic diagram based on silanization lipid probe separation excretion body.
Embodiment
Technical scheme is described in detail with reference to the accompanying drawings and examples.
Fig. 1 is the present invention using the schematic diagram based on silanization lipid probe separation excretion body.
Embodiment 1
1st, 35mm glass dishes plasma cleaner cleans 2 minutes, clean surface.
2nd, APTES-Macrogol 4000-phosphatidyl-ethanolamine (APTES-PEG4000-
DSPE) it is dissolved into ethanol solution and (needs matching while using) and is made into surface modification liquid.Lipid for separating excretion body is visited
The concentration of pin is 1 μm of ol.The glass dish being added to after cleaning.Glass dish needs at least to cover bottom.
3rd, lucifuge, it is incubated at room temperature 1 minute.Surface modification liquid is removed, with a large amount of pure water rinsings, removes the lipid being not bound with
Probe molecule.Dried after cleaning, baking oven toasts 1 minute.
4th, 1.5ml whole bloods are taken, 2500g is centrifuged 10 minutes at 4 DEG C, and it is standby to take out upper plasma.
5th, 800 μ l blood plasma are taken, are added in glass dish, 37 DEG C of shaking tables are incubated 2 minutes.
6th, blood plasma is removed, PBS is rinsed three times.Excretion body is adsorbed in the surface of lipid probe modification.
7th, after 0.1%SDS solution cracking excretion body, extraction nucleic acid, the nucleic acid amount for detecting to obtain using arms PCR methods,
The result that PCR is obtained shows that house-keeping gene circulates left and right can at 20 and enters increased logarithmic phase, shows this method separation
It is sufficient to obtain the nucleic acid that excretion body contains, can be used for other experimental analyses of follow-up test.
Embodiment 2
1st, 2ml vials plasma cleaner cleans 10 minutes, clean surface.
2nd, APTES-polyethylene glycol-800-1,2- dioleyl phosphatidyl cholines (APTES-
PEG800-DOPC) it is dissolved into ethanol solution and (needs matching while using) and is made into surface modification liquid.For separating excretion body
The concentration of lipid probe is 1pmol.It is added in the vial after cleaning.Vial adds more than 2/3rds.
3rd, lucifuge, it is incubated at room temperature 5 minutes.Surface modification liquid is removed, with a large amount of pure water rinsings, removes the lipid being not bound with
Probe molecule.Dried after cleaning, baking oven toasts 2 minutes.
4th, 1.5ml urines are taken, 2500g is centrifuged 10 minutes at 4 DEG C, and it is standby to take out upper strata.
5th, 800 μ l urine supernatants are taken, are added in vial, 37 DEG C of shaking tables are incubated 2 minutes.
6th, urine supernatant is removed, PBS is rinsed three times.Excretion body is adsorbed in the surface of lipid probe modification.
7th, excretion body is dyed using immobilized artificial membrane fluorescence probe DPH (10% ethanol solution), due to excretion body size
Too small, although single excretion body can not be observed directly with fluorescence microscope, fluorescence intensity ratio is not inhaled in general
The vial enhancing of attached excretion body.Illustrate that the excretion body that such a method obtains is applied to the experiment such as fluorescent staining, ELISA.
Embodiment 3
1st, 35mm glass dishes plasma cleaner cleans 5 minutes, clean surface.
2nd, APTES-PEG 8000-lysolecithin (APTES-PEG8000-Lyso
PC) it is dissolved into ethanol solution and (needs matching while using) and is made into surface modification liquid.For separating the lipid probe of excretion body
Concentration be 10nmol.It is added in the glass dish after cleaning.Glass dish needs at least to cover bottom.
3rd, lucifuge, it is incubated at room temperature 3 minutes.Surface modification liquid is removed, with a large amount of pure water rinsings, removes the lipid being not bound with
Probe molecule.Dried after cleaning, baking oven toasts 1 minute.
4th, 1.5ml salivas are taken, 2500g is centrifuged 10 minutes at 4 DEG C, and it is standby to take out upper strata.
5th, 800 μ l saliva supernatants are taken, are added in glass dish, 37 DEG C of shaking tables are incubated 2 minutes.
6th, desalivation supernatant is removed, PBS is rinsed three times.Excretion body is adsorbed in the surface of lipid probe modification.
7th, after 0.1%SDS solution cracking excretion body, protein is extracted, using Coomassie Brilliant Blue qualitative determination protein,
As a result show the presence of protein, illustrate the excretion body that such a method obtains, can be used for subsequent protein analysis etc. other
Experiment.
Claims (5)
- A kind of 1. method based on silanization lipid probe separation excretion body, it is characterised in that it is first by silane-PEG- lipids point Son is added in glassware so that silane-PEG- lipid moleculars by silicon oxygen bond and glassware surface silicon covalent bond, PEG- lipid moleculars modify glass surface；Then excretion body is added, PEG- lipid moleculars are inserted into the phospholipid bilayer of excretion body In layer, and then excretion body is fixed on glassware surface, realizes the separation of excretion body；Wherein：Silane-PEG- the lipids point The structure of son is shown below：Wherein：The lipid molecular is monoacyl lipid, any in diacyl lipid or cholesterol.
- 2. according to the method for claim 1, it is characterised in that contain in PE GIndividual oxirane EO.
- 3. according to the method for claim 1, it is characterised in that the carbochain of monoacyl lipid or diacyl lipid arrives in C10 Between C26.
- 4. according to the method for claim 1, it is characterised in that silane-PEG- lipid moleculars are APTES-PEG4000- DSPE, APTES-PEG800-DOPC or APTES-PEG8000-Lyso PC.
- 5. according to the method for claim 1, it is characterised in that comprise the following steps that：(1) glassware is cleaned using plasma cleaner cleaning 2-10 minutes to surface；(2) at room temperature, silane-PEG- lipid moleculars are dissolved into absolute ethyl alcohol, obtain concentration be 1pmol~1 μm ol silane- PEG- lipid solns；(3) silane-PEG- lipid solns are added in glassware, lucifuge, room temperature, are incubated 1~5 minute, discard decorating liquid, A large amount of pure water rinsings；(4) after glassware naturally dry, 55~65 DEG C of baking oven, toast 1~2 minute, it is standby；(5) fluid sample is added to after the modification that step (4) obtains in glassware, and 37 DEG C are incubated 2 minutes, and fluid sample includes Blood plasma, saliva and urine；(6) solution example is discarded, PBS is rinsed three times；Realize the separation of excretion body.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710579129.6A CN107365737A (en) | 2017-07-17 | 2017-07-17 | A kind of method based on silanization lipid probe separation excretion body |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710579129.6A CN107365737A (en) | 2017-07-17 | 2017-07-17 | A kind of method based on silanization lipid probe separation excretion body |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107365737A true CN107365737A (en) | 2017-11-21 |
Family
ID=60308371
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710579129.6A Pending CN107365737A (en) | 2017-07-17 | 2017-07-17 | A kind of method based on silanization lipid probe separation excretion body |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107365737A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897419A (en) * | 2021-11-08 | 2022-01-07 | 浙江大学 | Kit and method for extracellular vesicle capture or extracellular vesicle quantitative analysis or extracellular vesicle content quantitative analysis |
| CN116515844A (en) * | 2023-06-30 | 2023-08-01 | 四川大学华西医院 | Migration body aptamer and screening method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197066A (en) * | 2013-03-07 | 2013-07-10 | 美国纳米材料创新有限公司 | Immunoliposome biochip, preparation method thereof and application thereof in biological detection |
| US20160169876A1 (en) * | 2013-08-30 | 2016-06-16 | The University Of Tokyo | Exosome analysis method, exosome analysis chip, and exosome analysis device |
| CN106771206A (en) * | 2017-01-22 | 2017-05-31 | 胡家铭 | A kind of immunolipid polymer hybrid nanoparticle biochip and preparation method thereof and the application in disease detection |
-
2017
- 2017-07-17 CN CN201710579129.6A patent/CN107365737A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197066A (en) * | 2013-03-07 | 2013-07-10 | 美国纳米材料创新有限公司 | Immunoliposome biochip, preparation method thereof and application thereof in biological detection |
| US20160169876A1 (en) * | 2013-08-30 | 2016-06-16 | The University Of Tokyo | Exosome analysis method, exosome analysis chip, and exosome analysis device |
| CN106771206A (en) * | 2017-01-22 | 2017-05-31 | 胡家铭 | A kind of immunolipid polymer hybrid nanoparticle biochip and preparation method thereof and the application in disease detection |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897419A (en) * | 2021-11-08 | 2022-01-07 | 浙江大学 | Kit and method for extracellular vesicle capture or extracellular vesicle quantitative analysis or extracellular vesicle content quantitative analysis |
| CN116515844A (en) * | 2023-06-30 | 2023-08-01 | 四川大学华西医院 | Migration body aptamer and screening method and application thereof |
| CN116515844B (en) * | 2023-06-30 | 2023-09-08 | 四川大学华西医院 | Migration body aptamer and screening method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kang et al. | High-purity capture and release of circulating exosomes using an exosome-specific dual-patterned immunofiltration (ExoDIF) device | |
| CN107254430A (en) | A kind of method based on positive charge adsorbing separation excretion body | |
| Wu et al. | Biochemical analysis on microfluidic chips | |
| Hurwitz et al. | Extraction of extracellular vesicles from whole tissue | |
| Madara et al. | A simple approach to measurement of electrical parameters of cultured epithelial monolayers: use in assessing neutrophil-epithelial interactions | |
| CN106238110B (en) | Use filtering and sample transfer device isolation, accumulation, characterization and/or the method for determining microorganism | |
| EP3458854B1 (en) | Method and kit for capturing extracellular vesicles (evs) on a solid surface | |
| Hong et al. | Isolation of biologically active exosomes from plasma of patients with cancer | |
| CN107858324A (en) | A kind of method for the extracellular vesica including excretion body secreted based on anion exchange resin adsorbing separation cell to culture medium | |
| CN108795869A (en) | A kind of circulating tumor cell positive enrichment method | |
| CN110343664B (en) | Method for extracting exosome and exosome protein | |
| Santra et al. | Handbook of single cell technologies | |
| Kostal et al. | Recent advances in the analysis of biological particles by capillary electrophoresis | |
| CN206787889U (en) | A kind of device for separating and being enriched with body fluid components | |
| Shimasaki et al. | Exosome research and co-culture study | |
| CN109609458A (en) | A kind of method of low speed ultrafiltration centrifugation conjugated polymer sedimentation separation excretion body | |
| CN106244553A (en) | The separation of circulating tumor cell and detection method | |
| Raju et al. | Microfluidic platforms for the isolation and detection of exosomes: A brief review | |
| CN107365737A (en) | A kind of method based on silanization lipid probe separation excretion body | |
| CN108801746A (en) | A kind of device of separation and enrichment body fluid components | |
| CN104062428A (en) | Kit for detecting circulating tumor cells | |
| WO2021110939A1 (en) | Methods for identifying viral infections and for analyzing exosomes in liquid samples by raman spectroscopy | |
| Bojmar et al. | Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids | |
| WO2019129178A1 (en) | Composition containing anti-cd45 monoclonal antibody, and method for using same | |
| Kohl et al. | Isolation of outer membrane vesicles including their quantitative and qualitative analyses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171121 |
|
| RJ01 | Rejection of invention patent application after publication |