CN106906294A - A kind of quantitative approach of extracellular vesica/liposome - Google Patents
A kind of quantitative approach of extracellular vesica/liposome Download PDFInfo
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Abstract
The invention discloses a kind of quantitative approach of extracellular vesica/liposome.Anchoring molecule and nucleic acid are linked, then is incubated altogether with extracellular vesica/liposome, the compound is anchored on extracellular vesica/liposome；The unnecessary anchoring molecule nucleic acid complexes not linked with extracellular vesica/liposome are filtered out with super filter tube；To be linked with after the extracellular vesica/liposome of anchoring molecule nucleic acid complexes mixes with nucleic acid amplification mixed liquor carries out amplified reaction, quantitative.The present invention can realize the accurate absolute quantitation of extracellular vesica/liposome；The present invention is applied widely, can simultaneously provide specificity and non-specific two kinds of quantitative manners.
Description
Technical field
The invention belongs to biotechnology and field of nanometer material technology, more particularly to a kind of quantitative square of extracellular vesica/liposome
Method.
Background technology
Excretion body is a kind of nano vesicle with lipid bilayer Rotating fields of cell secretion, and particle diameter distribution is 30-
100nm, has highly important effect in intercellular mutual communication, can be used as biomarker is for the examination of disease and examines
It is disconnected.There is significant difference in the excretion body number that different cells or cell are secreted under different stressed conditions, therefore cell is secreted
The number of excretion body be important physiology, a pathological parameter.Therefore accurately quantitative analysis is carried out to excretion body in clinic
All have with scientific research field and be of great significance.But because the particle diameter of excretion body is too small, to its accurate quantification all face always
Face very big technical difficulty.
Conventional quantitative means have albuminimetry, nano particle trace analysis (NTA) technology, dynamic light scattering at present
(DLS) technology, qnano quantitative techniques, flow cytometer.
Albuminimetry refers to making relative quantitative assay to albumen contained by excretion body by the protein quantification such as BCA methods method
Connect the quantity for characterizing excretion body.Because this is a kind of indirectly relative quantitation method, its accuracy of quantitative analysis to excretion body is not
It is high.
Nano particle trace analysis (NTA) technology, the particle irradiated in suspension using laser, and caught with video camera
Scattering photoimaging produced by obtaining, and then the Brownian movement of individual particle is tracked, the concentration of particle is obtained, so as to realize to particle
Accurate metering.But realize needing sensitive imaging system, high cost to the tracking of individual particle.And reaction or reunion are drawn
The size for rising changes, protein masses can all influence measurement result, therefore sample needs thoroughly purifying.
Dynamic light scattering (DLS) technology is the particle for using laser to irradiate in suspension, determines the whole sample of different time
, there is correlation with particle concentration according to scattering strength in the change of scattering strength, based on series it is assumed that by scatter intensity distribution
It is converted into number distribution.But the initial information obtained because of DLS is scatter intensity distribution, it is necessary to be based on series it is assumed that ability
Scatter intensity distribution is converted into number distribution.This series of hypothesis and conversion can introduce larger error, and some react
Or particle size changes caused by reuniting, protein masses can also influence scattering strength, so as to forbidden the number of particles for obtaining
Really.
qnano：Under the driving of voltage or pressure, charged ion, by nano-pore, forms ion-conductance in the form of electrophoresis
Stream, measurable gas current reduction can be caused when charged particle to be measured migrates across nano-pore.According to nanoporous size and
Curent change number of times can obtain the particle diameter distribution and number of particles of particle.But there is non-spy between nano particle and nano-pore membrane
Opposite sex absorption, counts inaccurate.The size that reaction or reunion cause changes, protein masses can all influence measurement result, therefore
Sample needs thoroughly purifying.And also need to sensitively detect the sensor and the controllable nano-pore of size of gas current change
Film, high cost.
Flow cytometer：Generally, the actual grain size lower limit of commercial flow cytometer is about 300nm, and extracellular vesica or outer
Secrete body particle diameter universal under the numerical value, signal cannot make a distinction with baseline noise.Therefore need to be applicable the special type of small particle
Streaming plant, such as Apogee Flow Systems.But this special streaming plant, expensive, high cost.
The content of the invention
It is an object of the invention to overcome the shortcoming of prior art with it is not enough, there is provided an extracellular vesica/liposome is quantified
Method.Described method of counting can realize absolute quantitation, with low cost.
The purpose of the present invention is achieved through the following technical solutions：
The quantitative approach of extracellular vesica/liposome of the invention is comprised the following steps：
1) anchoring molecule and nucleic acid are linked；
2) anchoring molecule-nucleic acid complexes after connection are incubated altogether with extracellular vesica/liposome, by the compound grappling
Onto extracellular vesica/liposome；
3) the unnecessary anchoring molecule-nucleic acid complexes not linked with extracellular vesica/liposome are filtered out；
4) will be linked with after the extracellular vesica/liposome of anchoring molecule-nucleic acid complexes mixes with nucleic acid amplification mixed liquor
Amplified reaction is carried out, it is quantitative.
Extracellular vesica of the present invention includes excretion body and vesica.
Preferably, step 1) described in anchoring molecule be non-specific anchoring molecule or specific anchoring molecule, it is non-specific
Property anchoring molecule includes but is not limited to stearoyl (stearyl), cholesterol (cholesterol), glycosyl-phosphatidyl inositol
(glycosyl phosphatidyl inositol), oleyl chains (oleyl chain) and its derivative, preferably PEG modifications
Oleyl chains；Specific anchoring molecule includes but is not limited to antibody, aptamer, preferably aptamer.
When anchoring molecule is aptamer；Aptamer as anchoring molecule-nucleic acid complexes directly with it is extracellular
Vesica/liposome is incubated altogether, without linking with nucleic acid again.
Step 1) described in nucleic acid be single, double chain DNA or RNA, length 10Kb-200Kb；The preferably DNA of double-strand,
20Kb-50Kb；Described refers to be incited somebody to action by the chemical reaction between group in anchoring molecule and nucleic acid by anchoring molecule and nucleic acid link
The two links together, and preferably leads to the nucleic acid molecules of amino or carboxyl modified by carboxyl or amido modified anchoring molecule
The condensation reaction crossed between amino and carboxyl links together.
Step 2) described in common incubation refer to after anchoring molecule-nucleic acid complexes are mixed with extracellular vesica/liposome,
The 5min-24h times are incubated at 10 DEG C -80 DEG C, preferably 30 DEG C -50 DEG C, 10min-1h is incubated.
Step 3) described in filtering refer to super filter tube using 10-100KD molecular cut offs, in 4000-10000g conditions
Lower centrifugation 5-10min, is centrifuged 3-6 times.
Preferably, step 4) described in carry out amplified reaction including PCR amplification, nucleic acid isothermal amplification.
Preferably, step 4) in nucleic acid amplification mixed liquor refer to contain archaeal dna polymerase, primer, nucleic acid fluorescent probe
Archaeal dna polymerase, primer, nucleic acid fluorescent probe in mixed liquor, and mixed liquor are all relative to the number of extracellular vesica/liposome
Excessive.
Preferably, step 4) in quantitative approach include two classes, a class is to carry out relative quantification based on fluorescence signal intensity, separately
One class refers to that fluorescence signal point is counted to carry out absolute quantitation.
Described counting to fluorescence signal point carries out the method for absolute quantitation and refers to：Anchoring molecule-nucleic acid will be linked with multiple
Extracellular vesica/the liposome of compound is distributed in substantial amounts of drop or cell after mixing with nucleic acid amplification mixed liquor, carries out nucleic acid
Amplified reaction；Based on Poisson distribution, each drop or cell at most only have an extracellular vesica/liposome, and statistics has fluorescence to believe
Number drop or cell number, you can obtain the number of extracellular vesica/liposome.
Relative to prior art, the present invention has the advantages that:
1. the accurate absolute quantitation of extracellular vesica/liposome can be realized；
2. the raw material simplicity needed for realizing the present invention is easy to get, simple to operate, with low cost to equipment without particular/special requirement；
3. applied widely, specificity and non-specific two kinds of quantitative manners can be provided simultaneously.
Brief description of the drawings
Fig. 1 is the process chart that nucleic acid marking is carried out to a kind of extracellular vesica/liposome of the present invention；
Fig. 2 is the principle schematic that the digital pcr method absolute quantitation based on chip detects extracellular vesica/liposome；
Fig. 3 a are the transmission electron microscope picture of isolated excretion body from urine；
Fig. 3 b are the excretion body of nucleic acid marking through the fluorogram after nucleic acid amplification reaction；
Wherein label 1 mix with PCR mixed liquors for the excretion body of anchoring molecule-nucleic acid complexes after to carry out nucleic acid amplification anti-
The fluorescence that should be produced afterwards；
Label 2 is to carry out nucleic acid amplification reaction by the centrifugation waste liquid after 4 ultrafiltration, without generation fluorescence signal；
Label 3 is control group, and excretion body is replaced with into PBS carries out identical operating process, without generation fluorescence signal；
Fig. 4 carries out the quantitative fluorescence signal figure of excretion body for what is observed under light microscope using digital pcr chip；Its
Middle a is 10 times of Sample Dilution, and b is 1000 times of Sample Dilution.
Specific embodiment
Embodiment 1
The oleyl chains molecule of 100 μM of PEG modifications is mixed with 100 μM of nucleic acid, is reacted at room temperature overnight, will reacted
Mixed liquor afterwards is diluted to 10 μM.
It is as shown in Figure 3 a to be separated from urine using supercentrifugation using the isolated excretion body of supercentrifugation
The transmission electron microscope picture of the excretion body for arriving.
The mark flow of excretion body is as shown in Figure 1：The mixed liquor after certain volume dilution is taken to mix in equal volume with excretion body,
60min is reacted at being placed in 30 DEG C；By above-mentioned reaction solution add 100KD molecular cut offs super filter tube in, under 4000g rotating speeds from
Heart 10min, is centrifuged repeatedly 4 times, oleyl chains molecule and nucleic acid that elimination is not combined with excretion body.
Fig. 3 b show and process the excretion body for obtaining through the fluorogram after nucleic acid amplification by above-mentioned steps.Wherein label in figure
1 mix with PCR mixed liquors for the excretion body of anchoring molecule-nucleic acid complexes after carry out the fluorescence of generation after nucleic acid amplification reaction；
Label 2 is to carry out nucleic acid amplification reaction by the centrifugation waste liquid after 4 ultrafiltration, without generation fluorescence signal；Label 3 is control
Group, excretion body is replaced with into PBS carries out identical operating process, without generation fluorescence signal.Illustrate that nucleic acid is successfully connected to outer
Secrete on body, and left away completely by unreacted anchoring molecule-nucleic acid complexes after super filter tube centrifugation.
Above-mentioned centrifuged pellet is resuspended with 500 μ L pure water, take 10 μ L and draw with excessive nucleic acid polymerase, nucleic acid amplification
Thing, nucleic acid fluorescent probe probe, the mixing of nucleic acid amplification buffer solution, the μ L of cumulative volume 20 carry out real-time fluorescence quantitative PCR reaction.It is real
Now to the non-specific relative quantification of excretion body.
Embodiment 2
10 μM of aptamer molecules are mixed in equal volume with excretion body, 10min is reacted at being placed in 50 DEG C.
By in the super filter tube of above-mentioned reaction solution addition 50KD molecular cut offs, 5min is centrifuged under 10000g rotating speeds, repeatedly
Centrifugation 3 times, the aptamer molecule that elimination is not combined with excretion body.
Above-mentioned centrifuged pellet is resuspended with 500 μ L pure water, take 10 μ L and draw with excessive nucleic acid polymerase, nucleic acid amplification
Thing, nucleic acid fluorescent probe probe, the mixing of nucleic acid amplification buffer solution, the μ L of cumulative volume 20 carry out real-time fluorescence quantitative PCR reaction.It is real
Now to the specific relative quantification of excretion body.
Embodiment 3
The oleyl chains molecule of 100 μM of PEG modifications is mixed with 100 μM of nucleic acid, is reacted at room temperature overnight, will reacted
Mixed liquor afterwards is diluted to 10 μM.
The mixed liquor after certain volume dilution is taken again to mix in equal volume with excretion body, and 60min is reacted at being placed in 30 DEG C.
By in the super filter tube of above-mentioned reaction solution addition 100KD molecular cut offs, 5min is centrifuged under 6000g rotating speeds, repeatedly
Centrifugation 6 times, oleyl chains molecule and nucleic acid that elimination is not combined with excretion body.
Above-mentioned centrifuged pellet is resuspended with 500 μ L pure water, 100 μ L are taken with excessive nucleic acid polymerase, nucleic acid amplification
Primer, nucleic acid fluorescent probe probe, the mixing of nucleic acid amplification buffer solution, are settled to the μ L of cumulative volume 200.
Above-mentioned solution is encapsulated into a large amount of drops with droplet generator, carries out isothermal nucleic acid amplification reaction.
The drop for having fluorescence signal is counted using flow cytometer.It is definitely fixed to the non-specificity of excretion body to realize
Amount.
Embodiment 4
The oleyl chains molecule of 100 μM of PEG modifications is mixed with 100 μM of nucleic acid, is reacted at room temperature overnight, will reacted
Mixed liquor afterwards is diluted to 10 μM.
The mixed liquor after certain volume dilution is taken again to mix in equal volume with excretion body, and 60min is reacted at being placed in 30 DEG C.
By in the super filter tube of above-mentioned reaction solution addition 100KD molecular cut offs, 5min is centrifuged under 6000g rotating speeds, repeatedly
Centrifugation 6 times, oleyl chains molecule and nucleic acid that elimination is not combined with excretion body.
Above-mentioned centrifuged pellet is resuspended with 500 μ L pure water, 100 μ L are taken with excessive nucleic acid polymerase, nucleic acid amplification
Primer, nucleic acid fluorescent probe probe, the mixing of nucleic acid amplification buffer solution, are settled to the μ L of cumulative volume 200.
Above-mentioned solution is encapsulated into a large amount of drops with droplet generator, enters performing PCR reaction.
The drop for having fluorescence signal is counted using flow cytometer.It is definitely fixed to the non-specificity of excretion body to realize
Amount.
Embodiment 5
10 μM of aptamer molecules are mixed in equal volume with excretion body, 60min is reacted at being placed in 40 DEG C.
By in the super filter tube of above-mentioned reaction solution addition 100KD molecular cut offs, 10min is centrifuged under 5000g rotating speeds, repeatedly
Centrifugation 3 times, the aptamer molecule that elimination is not combined with excretion body.
Above-mentioned centrifuged pellet is resuspended with 500 μ L pure water, then with excessive nucleic acid polymerase, nucleic acid amplification primers,
Nucleic acid fluorescent probe probe, the mixing of nucleic acid amplification buffer solution, are settled to cumulative volume 1mL.
As shown in Fig. 2 above-mentioned mixing liquid is passed through into digital pcr chip, isothermal nucleic acid amplification reaction is carried out, in microscope
It is lower to count the cell number for having fluorescence signal.Realize the specific absolute quantitation to excretion body.
Embodiment 6
The oleyl chains molecule of 10 μM of PEG modifications is mixed in equal volume with excretion body, 60min is reacted at being placed in 50 DEG C.
By in the super filter tube of above-mentioned reaction solution addition 100KD molecular cut offs, 5min is centrifuged under 8000g rotating speeds, repeatedly
Centrifugation 5 times, the aptamer molecule that elimination is not combined with excretion body.
Above-mentioned centrifuged pellet is resuspended with 500 μ L pure water, then with excessive nucleic acid polymerase, nucleic acid amplification primers,
Nucleic acid fluorescent probe probe, the mixing of nucleic acid amplification buffer solution, are settled to cumulative volume for 1mL.Above-mentioned mixing liquid is passed through numeral
Pcr chip, enters performing PCR reaction, and statistics has the number of the cell of fluorescence signal under the microscope, as shown in Figure 4.A, b are respectively
10 times of the excretion body Sample Dilution of nucleic acid marking, it is passed through chip after 1000 times, the cell number that fluorescence is sent on chip is also presented
Corresponding multiple proportion.
Realize the specific absolute quantitation to excretion body.
Claims (10)
1. a kind of quantitative approach of extracellular vesica/liposome, it is characterised in that comprise the following steps：
1) anchoring molecule and nucleic acid are linked；
2) anchoring molecule-nucleic acid complexes after connection are incubated altogether with extracellular vesica/liposome, the compound is anchored into born of the same parents
On outer vesica/liposome；
3) the unnecessary anchoring molecule-nucleic acid complexes not linked with extracellular vesica/liposome are filtered out；
4) will be linked with after the extracellular vesica/liposome of anchoring molecule-nucleic acid complexes mixes with nucleic acid amplification mixed liquor is carried out
Amplified reaction, it is quantitative.
2. preparation method according to claim 1, it is characterised in that step 1) described in anchoring molecule be non-specific anchor
Determine molecule or specific anchoring molecule, non-specific anchoring molecule includes but is not limited to stearoyl, cholesterol, glycosyl-phosphatidyl flesh
Alcohol, oleyl chains and its derivative, specific anchoring molecule include but is not limited to antibody, aptamer.
3. preparation method according to claim 1, it is characterised in that when anchoring molecule is aptamer；Nucleic acid is adapted to
Body is directly incubated altogether as anchoring molecule-nucleic acid complexes with extracellular vesica/liposome, without linking with nucleic acid again.
4. preparation method according to claim 1, it is characterised in that step 1) described in nucleic acid be single, double chain DNA or
RNA, length 10Kb-200Kb；Described refers to by the change between group in anchoring molecule and nucleic acid by anchoring molecule and nucleic acid link
Learn reaction the two links together.
5. preparation method according to claim 1, it is characterised in that step 2) described in common incubation refer to by anchoring molecule-
After nucleic acid complexes mix with extracellular vesica/liposome, the 5min-24h times are incubated at 10 DEG C -80 DEG C.
6. preparation method according to claim 1, it is characterised in that step 3) described in filtering refer to use 10-100KD
Molecular cut off, is centrifuged 5-10min under the conditions of 4000-10000g, is centrifuged 3-6 times.
7. preparation method according to claim 1, it is characterised in that step 4) described in carry out amplified reaction include it is poly-
Polymerase chain reaction amplification, nucleic acid isothermal amplification.
8. preparation method according to claim 1, it is characterised in that step 4) in nucleic acid amplification mixed liquor refer to containing
Archaeal dna polymerase, primer, the mixed liquor of nucleic acid fluorescent probe, each material in mixed liquor is relative to extracellular vesica/liposome
Number be all excessive.
9. preparation method according to claim 1, it is characterised in that step 4) in quantitative approach include two classes, a class is base
Relative quantification is carried out in fluorescence signal intensity, another kind of refers to that fluorescence signal point is counted to carry out absolute quantitation.
10. preparation method according to claim 9, it is characterised in that it is definitely fixed that described being counted to fluorescence signal point is carried out
The method of amount refers to：Extracellular vesica/liposome that anchoring molecule-nucleic acid complexes will be linked with mixes with nucleic acid amplification mixed liquor
After be distributed in substantial amounts of drop or cell, carry out nucleic acid amplification reaction；Based on Poisson distribution, each drop or cell at most have
One extracellular vesica/liposome, statistics has the drop or cell number of fluorescence signal, you can obtain extracellular vesica/liposome
Number.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107870244A (en) * | 2017-11-02 | 2018-04-03 | 蔡慧娜 | A kind of immunoliposome LAMP method for the super quick detection of protein |
| CN109576207A (en) * | 2018-12-17 | 2019-04-05 | 贺川江 | A kind of separation method of the extracellular vesica based on metal ion network mixture |
| CN110106233A (en) * | 2019-03-25 | 2019-08-09 | 杭州师范大学 | A kind of digital pcr detection method of extracellular vesica/excretion body |
| CN110229820A (en) * | 2019-06-17 | 2019-09-13 | 南京鼓楼医院 | The special aptamers screening technique of extracellular vesicle surface albumen based on partial size back-and-forth method |
| CN110724734A (en) * | 2019-10-22 | 2020-01-24 | 北京恩泽康泰生物科技有限公司 | Artificial liposome containing miRNA micic and preparation method and application thereof |
| WO2020029123A1 (en) * | 2018-08-08 | 2020-02-13 | 深圳宣泽生物医药有限公司 | Method for modifying extracellular vesicles |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107870244A (en) * | 2017-11-02 | 2018-04-03 | 蔡慧娜 | A kind of immunoliposome LAMP method for the super quick detection of protein |
| WO2020029123A1 (en) * | 2018-08-08 | 2020-02-13 | 深圳宣泽生物医药有限公司 | Method for modifying extracellular vesicles |
| CN109576207A (en) * | 2018-12-17 | 2019-04-05 | 贺川江 | A kind of separation method of the extracellular vesica based on metal ion network mixture |
| CN110106233A (en) * | 2019-03-25 | 2019-08-09 | 杭州师范大学 | A kind of digital pcr detection method of extracellular vesica/excretion body |
| CN110229820A (en) * | 2019-06-17 | 2019-09-13 | 南京鼓楼医院 | The special aptamers screening technique of extracellular vesicle surface albumen based on partial size back-and-forth method |
| CN110724734A (en) * | 2019-10-22 | 2020-01-24 | 北京恩泽康泰生物科技有限公司 | Artificial liposome containing miRNA micic and preparation method and application thereof |
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