CN109082400A - A method of excretion body being separated from biological sample using DEAE magnetic nano particle - Google Patents
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Abstract
The invention discloses a kind of methods for separating excretion body from biological sample using DEAE magnetic nano particle.The method that the present invention provides a kind of to separate excretion body from biological sample is that excretion body is separated from biological sample using DEAE magnetic nano particle.The partial size of the DEAE magnetic nano particle is 200-30000nm.Described method includes following steps: (1) excretion body is captured from biological sample using DEAE magnetic nano particle；(2) using the excretion body on eluent separation DEAE magnetic nano particle.The excretion body of expeditiously separating high-purity may be implemented in method provided by the invention, then carries out albumen and foranalysis of nucleic acids to the excretion body of capture, further using in biological detection or medical inspection.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of to be separated from biological sample using DEAE magnetic nano particle
The method of excretion body.
Background technique
Excretion body be a kind of cell secrete to extracellular diameter 30-150nm film property vesica.Cell is made by endocytosis
Formation vesicles intracellular are taken in by extracellular substance, then, vesicles occur fusion and form early endosome and further drill
Become late endosome.Intracytoplasmic some albumen and miRNA enter late endosome and then evolve into multivesicular body.Vesicles
It is released to extracellular to form excretion body.Have in cell culture medium and body fluid (blood plasma, serum, urine, saliva etc.) outer
Secrete the presence of body.The outer membrane of excretion body is phospholipid bilayer, and contains some functional proteins.In excretion body containing protein,
Lipid, mRNA, miRNA etc..According to the literature, excretion body wide participation cell communication, immune response, metastases waited
Journey.The excretion body of tumors secrete contains the albumen, microRNA, long-chain non-coding RNA of tumour-specific, therefore can pass through inspection
The excretion body of tumors secrete is surveyed to realize the early diagnosis of cancer.The fast and efficiently extraction separation for wanting excretion body is excretion
The basis of body research.
Existing excretion body separating and extracting process mainly has a ultracentrifugation, immunization, ultrafiltration, commercial kit,
Chromatographic exclusion etc..Ultracentrifugation first passes through differential centrifugation and removes cell and fragment, and then ultracentrifugation (16000rpm) is rich
Collect excretion body.Ultracentrifugation method generally requires 6-8 hours, and the rate of recovery is relatively low, only 5-25%, somewhat expensive.Immunization
Excretion body is mainly captured using the magnetic bead of antibody modification, obtained excretion body high-purity, rate of recovery height.But universality is low,
Only excretion body of the capture containing target protein, be easy to cause the analysis of distortion.Ultrafiltration is to be made using the method vacuumized containing outer
The solution of body is secreted by microfiltration membranes, so that excretion body is enriched in microfiltration membranes, this method bioaccumulation efficiency is relatively high, but easily
In blocking, and excretion knows from experience the damage that is under pressure.It there is now some commercialized kits (such as ExoQuick, Total
Exosome Isolation) extract excretion body, it is easy to use, but operation overnight is needed, and operating environment is open, it may
There is chemical agent residue to influence the operation such as subsequent qPCR, sequencing.These many conventional excretion body isolation technics all have centainly
Limitation.
Summary of the invention
The object of the present invention is to provide a kind of methods for separating excretion body from biological sample using DEAE magnetic nano particle.
The method that the present invention provides a kind of to separate excretion body from biological sample is to use DEAE magnetic nano particle from life
Excretion body is separated in object sample.
The partial size of the DEAE magnetic nano particle is 200-30000nm.
The partial size of the DEAE magnetic nano particle concretely 200nm.
Described method includes following steps:
(1) excretion body is captured from biological sample using DEAE magnetic nano particle；
(2) using the excretion body on eluent separation DEAE magnetic nano particle；
The eluent is salting liquid, and/or, the pH value of the eluent is 6-9.
The eluent is the aqueous solution containing 100-500mMNaCl and 50mM HEPES.
Aqueous solution of the eluent concretely containing 200mMNaCl and 50mM HEPES.
The pH of the eluent concretely 7.2.
In the step (1), the concentration of DEAE magnetic nano particle is 0.5-3mg/mL,
And/or the incubation time of DEAE magnetic nano particle and biological sample is 5-30min.
In the step (1), the concentration of DEAE magnetic nano particle is specially 5mg/mL,
And/or the incubation time of DEAE magnetic nano particle and biological sample is specially 10min.
The incubation time of the eluent and magnetic nano particle is 1min.
After the step (1), further include the steps that cleaning impurity using cleaning solution；
The cleaning solution is the aqueous solution containing 25-50mMNaCl and 50mM HEPES.
The pH of the cleaning solution is 6.2.
When the biological sample is blood plasma, water of the cleaning solution concretely containing 50mMNaCl and 50mM HEPES
Solution.
When the biological sample is urine, water of the cleaning solution concretely containing 25mMNaCl and 50mM HEPES
Solution.
Before the step (1), further include the steps that balancing DEAE magnetic nano particle using equilibrium liquid；
The equilibrium liquid is the aqueous solution containing 25mMNaCl and 50mM HEPES.
The pH of the equilibrium liquid is 6.2.
The equilibration time is 2min.
The present invention also protects application of the DEAE magnetic nano particle in separation excretion body.
The present invention also protects a kind of for separating the kit of excretion body, including DEAE magnetic nano particle and any of the above institute
The eluent stated.The kit further includes the cleaning solution of any description above.The kit further includes any description above
Equilibrium liquid.
The present invention also protects application of any description above method in biological detection or medical inspection.
Any description above DEAE magnetic nano particle is the magnetic nano particle for being coated with DEAE.The DEAE magnetic nanometer
The magnetic kernel that particle is concretely made of metal oxide and the high molecular polymer (DEAE- being wrapped in outside magnetic kernel
Cellulose) composition magnetic nano particle.The metal oxide concretely magnetic iron oxide.The DEAE magnetic nano particle is more
The concretely fluidMAG-DEAE of Chemicell company.
Any description above biological sample can be cell culture medium, blood, blood plasma, urine, milk, saliva, milk etc..
Present invention firstly provides a kind of utilization diethyl amino ethyl group (DEAE) magnetic nano particles to realize from biological sample outside
Secrete the method for body separation.Firstly, being balanced with equilibrium liquid to DEAE magnetic nano particle, then received with the DEAE magnetic of suitable concentration
Rice grain captures excretion body from biological sample, is eluted with the salting liquid of gradient concentration, and eluent is collected, with NTA, detection
The purity of excretion bulk concentration and excretion body that the salting liquid of various concentration affords, filters out optimal salinity, then
Optimal salinity and pH is selected to separate excretion body, to realize the excretion body of expeditiously separating high-purity.DEAE magnetic is received
Difference of the rice grain based on the electrification property such as excretion body and protein nucleic acid, the method with anion exchange are isolating and purifying excretion
Body.In principle, DEAE magnetic nano particle can capture all excretion bodies.In this patent, inventor utilizes DEAE magnetic nanometer
Particle realizes in 30min and efficiently isolates and purifies excretion body, and the rate of recovery of excretion body is up to 90%, is higher than existing hypervelocity
The methods of centrifugation, immunomagnetic beads, the time for greatly reducing the separation of excretion body, (ultracentrifugation needed 6-8h, immunomagnetic beads method
Need 3h or more).Since DEAE magnetic nano particle method is without the reagent that can destroy excretion body, isolated excretion body is complete
Whole property is fine.In addition, the versatility of DEAE magnetic nano particle method is fine, can be not only used for separating excretion body from blood plasma, also
It can be used for separating excretion body from urine, cell culture medium, provide good tool for the quick separating detection of excretion body.
Detailed description of the invention
Fig. 1 is the flow chart for separating excretion body from blood plasma using DEAE magnetic nano particle.
Fig. 2 is excretion body qualification result.A:TEM observes the form of the excretion body of DEAE magnetic nano particle separation；B:NTA is surveyed
The particle diameter distribution of the excretion body of DEAE magnetic nano particle separation.
Fig. 3 is excretion body elution efficiency and Tot Prot statistical result.
Fig. 4 is the CD81 concentration statistical result in excretion body.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Diethyl amino ethyl group (DEAE) magnetic nano particle: Chemicell company, fluidMAG-DEAE, partial size: 200nm；It is dense
Degree: 25mg/mL is stored in ddH2In O.
Embodiment 1 isolates and purifies excretion body using DEAE magnetic nano particle from blood plasma
This method flow chart is shown in Fig. 1, specific as follows:
One, DEAE magnetic nano particle is balanced
1,100 μ LDEAE magnetic nano particle vortex oscillation 30s are taken, DEAE magnetic nano particle is resuspended sufficiently, obtains magnetic and receives
Rice grain suspension.
2, it after completing step 1, takes 100 μ L magnetic nano particle suspensions to be placed in 1mL EP pipe, EP pipe is placed on magnetic frame,
Magnetic Isolation is carried out to magnetic nano particle suspension, abandons supernatant, is added 25mMNaCl+50mM HEPES aqueous solution (pH=6.2)
Magnetic nano particle is resuspended, 2min is balanced to the positive charge on DEAE magnetic nano particle surface.
3, after completing step 2, DEAE magnetic nano particle is drawn to EP tube bottom with magnetic frame, careful inhale abandons supernatant, collects magnetic
Nano particle.
Two, blood plasma pre-processes
The fresh whole blood for taking 10mL in vitro, 3000g, 4 DEG C of centrifugation 10min remove haemocyte, careful to take out upper layer blood
Slurry, every pipe 1mL packing are spare.
Three, DEAE magnetic nano particle captures excretion body from blood plasma
1, the blood plasma that 1ml step 2 obtains is mixed into (magnetic nano particle with the magnetic nano particle after step 1 Balance Treatment
Concentration be 0.5-3mg/mL), be incubated for a period of time (5-30min) combine excretion body and magnetic nano particle sufficiently.
2, after completing step 1, the magnetic nano particle for combining excretion body is drawn to EP tube bottom with magnetic frame, supernatant is abandoned, adds
Enter 50mMNaCl+50mM HEPES aqueous solution (pH=6.2), washes positively charged albumen and uncharged large biological molecule
Equal impurity, then adsorb magnetic nano particle with magnetic frame, collect supernatant and magnetic nano particle respectively.
3, the supernatant that step 2 is collected and the blood plasma that step 2 obtains PBS are diluted 50 times respectively, uses magnetic nano particle
Follow-up analysis instrument (NTA) detects the excretion bulk concentration in supernatant and primitive plasma, calculates capture rate.
Capture rate (%)=(1-A/B) × 100%；
A: the excretion bulk concentration after capture excretion body in supernatant；B: the excretion bulk concentration in blood plasma.
By optimization, the best magnetic nano particle concentration for capturing excretion body in step 1 from blood plasma is 5mg/mL, from blood plasma
The best incubation time of middle capture excretion body is 10min, and capture rate is up to 90%.
Four, excretion body is eluted
NaCl (100,200,300,400 and the 500mM)+50mM HEPES aqueous solution (pH=of various concentration is used respectively
7.2) magnetic nano particle collected with the 2 of step 3 is incubated for 1min, collects eluent, obtains excretion body suspension, excretion body is hanged
Liquid dilutes 50 times, surveys the excretion bulk concentration in eluent with magnetic nano particle follow-up analysis instrument (NTA).
Calculate the elution efficiency of excretion body, elution efficiency (%)=(1-C/D) × 100%；
C: the excretion bulk concentration after elution in supernatant；D: the excretion bulk concentration in blood plasma.
As a result as shown in Figure 3.The result shows that when the concentration of NaCl is 200mM in eluent, the elution efficiency of excretion body >=
90%.
Five, TEM identifies excretion body
The excretion body suspension that 7 μ L step 4 are obtained is added dropwise on 400 mesh copper mesh, is incubated at room temperature 1min, with filter paper from side
Face carefully sucks supernatant, and 7 μ L acetic acid uranium are then added dropwise, and is incubated at room temperature 15s, carefully sucks supernatant from side with filter paper, repeats 2
Secondary, copper mesh is placed to 2min at room temperature dries it completely.Electric microscopic observation excretion volume morphing is projected, is as a result seeing Fig. 2A,
Typical " saucer shape " structure is presented in excretion body.
Six, NTA identifies excretion body
The excretion body suspension PBS that step 4 is afforded dilutes 50 times, with magnetic nano particle follow-up analysis instrument (NTA)
Survey excretion body particle diameter distribution and the concentration in above-mentioned eluent.As a result see Fig. 2 B.The result shows that the particle diameter distribution of excretion body, outside
It is very uniform to secrete body particle diameter distribution, average grain diameter 120nm.
Embodiment 2 captures excretion body using DEAE magnetic nano particle from urine
One, DEAE magnetic nano particle is balanced
1,100 μ LDEAE magnetic nano particle vortex oscillation 30s are taken, DEAE magnetic nano particle is resuspended sufficiently, obtains magnetic and receives
Rice grain suspension.
2, it after completing step 1, takes 100 μ L magnetic nano particle suspensions to be placed in 1mL EP pipe, EP pipe is placed on magnetic frame,
Magnetic Isolation is carried out to magnetic nano particle suspension, abandons supernatant, is added 25mMNaCl+50mM HEPES aqueous solution (pH=6.2)
Magnetic nano particle is resuspended, 2min is balanced to the positive charge on DEAE magnetic nano particle surface.
3, after completing step 2, DEAE magnetic nano particle is drawn to EP tube bottom with magnetic frame, careful inhale abandons supernatant, collects magnetic
Nano particle.
Two, urine pre-processes
10ml freshly voided urine is taken, 3000g, 4 DEG C of centrifugation 10min, removal urine residue is careful to take out the clear urine in upper layer,
Every pipe 1mL packing is spare.
Three, DEAE magnetic nano particle captures excretion body from urine
1, the urine that 1ml step 2 obtains is mixed into (magnetic nano particle with the magnetic nano particle after step 1 Balance Treatment
Concentration be 5mg/mL), be incubated for 10min combine excretion body and magnetic nano particle sufficiently.
2, after completing step 1, the magnetic nano particle for combining excretion body is drawn to EP tube bottom with magnetic frame, abandons supernatant,
It is added 25mM NaCl+50mM HEPES aqueous solution (pH=6.2), washes positively charged albumen and uncharged biology is big
Then the impurity such as molecule adsorb magnetic nano particle with magnetic frame, abandon supernatant.
Four, excretion body is eluted
It is incubated for 10min with the NaCl solution (pH=7.2) and magnetic nano particle of 50mM HEPES+200mM, collects elution
Liquid obtains excretion body suspension.
Five, ELISA surveys the concentration of excretion body marker CD81
1, respectively add the 50 μ L of standard items of various concentration in ELISA standard sample sample wells；Step is separately added into sample to be tested hole
Each 10 μ L of excretion body (N=3) that the excretion body (N=3) and ultracentrifugation that rapid four DEAE magnetic nano particle extracts extract, then
Add 40 μ L of sample diluting liquid.
Ultracentrifugation extraction step is as follows: (1) taking 1mL urine, 3000g is centrifuged 10min, takes supernatant；(2) supernatant 160,
000g, 4 DEG C of centrifugation 70min abandon supernatant, collect precipitating；(3) it is resuspended and is precipitated with PBS, 160,000g, 4 DEG C of centrifugation 70min are collected
Precipitating；(4) precipitating is resuspended with 100 μ L PBS and obtains excretion body.
2, after completing step 1, every hole adds detection 100 μ L of primary antibody, seals reacting hole, 37 DEG C of incubation 60min with sealing plate film.
3, after completing step 2, liquid is discarded, every hole adds 200 μ L cleaning solutions, stands 1min, cleaning solution is got rid of, on blotting paper
It pats dry, is repeated 5 times.
4, after completing step 3, each 50 μ L of reaction substrate A, B is added in every hole, and 37 DEG C are protected from light incubation 15min.50 μ L are added to terminate
Liquid terminates digestion, surveys each hole in the absorbance of 450nm using microplate reader.Calculate excretion bulk concentration.
Reagent used in above-mentioned steps is all from CD81ELISA kit (FreeMore).
As a result as shown in Figure 4.The result shows that the excretion bulk concentration that DEAE magnetic nano particle extracts is higher, it is ultracentrifugation
Twice of the excretion bulk concentration of extraction.
Claims (10)
1. a kind of method for separating excretion body from biological sample is outside being separated from biological sample using DEAE magnetic nano particle
Secrete body.
2. the method as described in claim 1, it is characterised in that: the partial size of the DEAE magnetic nano particle is 200-30000nm.
3. method according to claim 1 or 2, it is characterised in that: described method includes following steps:
(1) excretion body is captured from biological sample using DEAE magnetic nano particle；
(2) using the excretion body on eluent separation DEAE magnetic nano particle；
The eluent is salting liquid, and/or, the pH value of the eluent is 6-9.
4. method as claimed in claim 3, it is characterised in that: the eluent is to contain 100-500mMNaCl and 50mM
The aqueous solution of HEPES.
5. the method as claimed in claim 3 or 4, it is characterised in that:
In the step (1), the concentration of DEAE magnetic nano particle is 0.5-3mg/mL,
And/or the incubation time of DEAE magnetic nano particle and biological sample is 5-30min.
6. method as claimed in claim 5, it is characterised in that:
In the step (1), the concentration of DEAE magnetic nano particle is 5mg/mL,
And/or the incubation time of DEAE magnetic nano particle and biological sample is 10min.
7. the method as described in claim 3 to 6 is any, it is characterised in that:
After the step (1), further include the steps that cleaning impurity using cleaning solution；
The cleaning solution is the aqueous solution containing 25-50mMNaCl and 50mM HEPES.
Application of the 8.DEAE magnetic nano particle in separation excretion body.
9. it is a kind of for separating the kit of excretion body, including elution described in DEAE magnetic nano particle and claim 3 or 4
Liquid.
10. application of any method of claim 1 to 7 in biological detection or medical inspection.
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Application publication date: 20181225 |
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