CN109116010A - Test tube and excretion body separation method for the acquisition of blood excretion body - Google Patents
Test tube and excretion body separation method for the acquisition of blood excretion body Download PDFInfo
- Publication number
- CN109116010A CN109116010A CN201810963224.0A CN201810963224A CN109116010A CN 109116010 A CN109116010 A CN 109116010A CN 201810963224 A CN201810963224 A CN 201810963224A CN 109116010 A CN109116010 A CN 109116010A
- Authority
- CN
- China
- Prior art keywords
- test tube
- excretion body
- blood
- adsorption structure
- acquisition
- 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.)
- Granted
Links
- 238000012360 testing method Methods 0.000 title claims abstract description 122
- 230000029142 excretion Effects 0.000 title claims abstract description 77
- 210000004369 blood Anatomy 0.000 title claims abstract description 30
- 239000008280 blood Substances 0.000 title claims abstract description 30
- 238000000926 separation method Methods 0.000 title claims abstract description 12
- 238000001179 sorption measurement Methods 0.000 claims abstract description 55
- 229920000669 heparin Polymers 0.000 claims abstract description 44
- 229960002897 heparin Drugs 0.000 claims abstract description 44
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 39
- 239000000463 material Substances 0.000 claims abstract description 21
- 238000010276 construction Methods 0.000 claims abstract description 13
- 210000000813 small intestine Anatomy 0.000 claims abstract description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 11
- 150000007523 nucleic acids Chemical class 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 11
- 210000000601 blood cell Anatomy 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 8
- 210000004027 cell Anatomy 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- -1 polyethylene Polymers 0.000 claims description 6
- 239000004698 Polyethylene Substances 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 5
- 238000005336 cracking Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 238000000159 protein binding assay Methods 0.000 claims description 5
- 239000004615 ingredient Substances 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000004743 Polypropylene Substances 0.000 claims description 3
- 238000010494 dissociation reaction Methods 0.000 claims description 3
- 230000005593 dissociations Effects 0.000 claims description 3
- 229920000573 polyethylene Polymers 0.000 claims description 3
- 229920001155 polypropylene Polymers 0.000 claims description 3
- 229920002635 polyurethane Polymers 0.000 claims description 3
- 239000004814 polyurethane Substances 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 230000007062 hydrolysis Effects 0.000 claims 1
- 238000006460 hydrolysis reaction Methods 0.000 claims 1
- 238000000338 in vitro Methods 0.000 claims 1
- 210000004185 liver Anatomy 0.000 claims 1
- 229920002521 macromolecule Polymers 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 10
- 238000013461 design Methods 0.000 abstract description 3
- 241000973497 Siphonognathus argyrophanes Species 0.000 description 12
- 238000005199 ultracentrifugation Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 238000011020 pilot scale process Methods 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5082—Test tubes per se
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Abstract
The present invention relates to molecular biology and clinical testing techniques fields, specifically a kind of haemocyte without being centrifuged in removal sample, it is structurally reasonable, test tube and excretion body separation method for the acquisition of blood excretion body easily to operate, equipped with test tube main body, it is characterized in that inboard wall of test tube has the adsorption structure for increasing 10 times or more using specific surface area made of heparin modified high molecular material, adsorption structure is arranged in test tube lower part, the adsorption structure is porous structure or tooth array or Fractal array adsorption structure or class small intestine wall construction, the present invention is compared with the prior art and design, facilitate with acquisition timely, it is structurally reasonable, the significant advantage such as easy to operate.
Description
Technical field:
It is specifically a kind of without being centrifuged removal sample the present invention relates to molecular biology and clinical testing techniques field
In haemocyte, it is structurally reasonable, easily to operate for blood excretion body acquisition test tube and excretion body separation method.
Background technique:
Excretion body is the vesicles that diameter is 30-150nm, is selectively packed and is discharged by living cells.In the body of human body
In liquid, such as blood, urine and cerebrospinal fluid, rich content.Contain different types of lipid, nucleic acid and albumen in excretion body
Deng these substances can be transported to specific target cell, to play corresponding biological function.Therefore, excretion body is thin
Huge effect is played in Intercellular communication and physiology and pathologic process.
Currently, there are mainly three types of the most common extracting methods of serum excretion body, one is ultracentrifugation method, the second is,
Immunomagnetic beads method, the third is RNA isolation kit, the relevant serum excretion body provided such as the Thermo company in the U.S. and SBI company
Reagent preparation.But three has clearly disadvantageous place, ultracentrifugation method, excretion body purity is high obtained, but produces
Rate is low, and time-consuming, complicated for operation, and expensive equipment is not suitable for hospital's detection practice, and repeated centrifugation operation is it is also possible to vesica
It damages, to reduce its quality；Immunomagnetic beads method, be capable of specificity captures corresponding excretion body, but this skill
Art antibody higher cost, only a small number of Zoomlions company is grasped, such as SBI company of the U.S., is caused with high costs；Sedimentation examination
The yield that agent box method extracts excretion body is high, but its purity is too low, while obtaining excretion body, can also obtain in many serum
The protein of high abundance.In addition, existing excretion body isolation technics usually after infecting a period of time using Western blot come
Verifying, is unable to real-time monitoring.
Summary of the invention:
The present invention is directed to shortcoming and defect existing in the prior art, propose it is a kind of without centrifugal treating, it is structurally reasonable,
Test tube and excretion body separation method for the acquisition of blood excretion body easily to operate.
The present invention can be achieved by the following measures:
A kind of test tube for the acquisition of blood excretion body, is equipped with test tube main body, it is characterised in that inboard wall of test tube, which has, to be used
Specific surface area made of heparin modified high molecular material increases 10 times or more of adsorption structure, and adsorption structure is arranged under test tube
Portion.
Adsorption structure of the present invention is porous structure or tooth array or Fractal array adsorption structure or class intestinal wall knot
Structure.
The groove structure convenient for accelerating the discharge of blood blood cell is additionally provided in test tube of the present invention, the setting of the groove structure exists
The top of adsorption structure, is equipped with the two or more grooves being arranged around inboard wall of test tube in groove structure, groove along test tube central axes by
The top of adsorption structure extends to test tube mouth.
The 6-16 V-groove that groove structure of the present invention is uniformly arranged around inboard wall of test tube, between adjacent groove
Boss surface is convexly curved, convenient for accelerating blood or blood cell discharge.
Test tube main body of the present invention is integrally formed using heparin modified high molecular material, or using by heparin modified high score
Embedded type structure made of sub- material be embedded in common test tube in, the heparin modified high molecular material be heparin modified polyethylene or
Heparin modified polypropylene or heparin modified PVC or heparin modified polyurethane or heparin modified PVA.
There is smooth bottom surface in test tube of the present invention, be discharged convenient for blood.
The present invention is equipped with annular outer platform in order to hold and handle, in test tube mouth outer wall, and pipe close is equipped on test tube mouth.
Class small intestine wall construction in adsorption structure of the present invention refers to that inboard wall of test tube is equipped with specific surface area and increases 10 times
Above policae circulane, pleat are fold.
The cone cell teeth that tooth array in adsorption structure of the present invention is spaced by altitude range for 0.5-2 millimeters, by that
This spacing range is that the strip groove structural arrangement of 1-3 millimeters deep forms, and wherein ditch piston ring land is equipped with extra projection or groove to increase
Add surface area；Wherein tooth array is formed by sawtooth unit repeated arrangement, and the sawtooth unit is successively arranged 3-6 from top to bottom
The different V-type tooth of length, the length of 3-6 V-type tooth are gradually reduced from top to bottom.
Pore diameter range is 0.05-0.5mm in porous structure in adsorption structure of the present invention, hole depth range is
0.05-0.1mm, aperture shape are circular hole or slotted eye or square hole or elongate holes or tri-angle-holed or pentagon hole or six
Side shape hole.
The invention also provides a kind of excretion body separation methods using above-mentioned test tube, it is characterised in that including following step
It is rapid:
Step 1: taking above-mentioned test tube, blood to be processed is added in test tube, additional amount is 5-10 milliliters；Step 2: shaking examination
Pipe, comes into full contact with sample in test tube with the adsorption structure on inboard wall of test tube；Step 3: pouring step 2 treated examination intraluminal fluid
Body, the excretion somatocyst bubble substance in sample is attached on the adsorption structure on inboard wall of test tube at this time；
Step 4: tube wall is carefully cleaned multiple times with phosphate buffer
Step 5: appropriate excretion body cracking is added to test tube in step 4 and RNA extracts reagent trizol, or heparin is added
Hydrolase or 8 mol/L sodium chloride solutions dissociation excretion body carry out analysis of protein；
Step 5: being settled by nucleic acid and obtain nucleic acid substances, or pass through antibody binding assay protein ingredient and content.
The present invention there is acquisition to facilitate timely compared with the prior art and design, structurally reasonable, easy to operate etc. significant
Advantage.
Detailed description of the invention:
Attached drawing 1 is structural schematic diagram of the invention.
Attached drawing 2 is the sectional view of groove structure in Fig. 1.
Attached drawing 3 is the schematic diagram of adsorption structure in the present invention.
Attached drawing 4 is a kind of embodiment structure schematic diagram of tooth array in the present invention.
Attached drawing 5 is to separate excretion body excretion body grain size distribution obtained using conventional centrifugal method in embodiment 3.
Attached drawing 6 is in embodiment 3 using cuvette construction excretion body grain size distribution obtained in embodiment 1.
Attached drawing 7 is in embodiment 3 using cuvette construction excretion body grain size distribution obtained in embodiment 2.
Attached drawing 8 is in embodiment 3 using conventional centrifugal method separation excretion body excretion bulk concentration FITC-SS two dimension obtained
Scatter plot.
Attached drawing 9 is in embodiment 3 using cuvette construction separation excretion body excretion bulk concentration obtained in embodiment 1
FITC-SS two dimension scatter plot.
Attached drawing 10 is in embodiment 3 using cuvette construction separation excretion body excretion bulk concentration obtained in embodiment 2
FITC-SS two dimension scatter plot.
Appended drawing reference: test tube main body 1, adsorption structure 2, bottom surface 3, groove structure 4, annular outer platform 5, groove 6.
Specific embodiment:
The present invention is further illustrated with reference to the accompanying drawings and examples.
As shown in the picture, the invention proposes a kind of test tubes for the acquisition of blood excretion body, are equipped with test tube main body, special
Sign is that inboard wall of test tube has the adsorption structure for increasing 10 times or more using specific surface area made of heparin modified high molecular material,
The adsorption structure is porous structure or tooth array or FRACTAL ADSORPTION structure or class small intestine wall construction, is had in test tube smooth
Bottom surface 3, adsorption structure 2 are arranged in test tube lower part, are additionally provided with as shown in Fig. 2, in test tube convenient for accelerating the discharge of blood blood cell
Groove structure 4, the top that adsorption structure 2 is arranged in of the groove structure 4 are equipped with two or more in groove structure 4 around test tube
The groove 6 of inner wall setting, groove 6 extend to test tube mouth by the top of adsorption structure along test tube central axes.
6 or 8 V-grooves 6 that groove structure 4 of the present invention is preferably uniformly arranged around inboard wall of test tube, adjacent groove 6
Between boss surface be convexly curved, convenient for accelerate blood or blood cell discharge.
Test tube main body 1 of the present invention is integrally formed using heparin modified high molecular material, or using by heparin modified height
Embedded type structure made of molecular material is embedded in common test tube, and the heparin modified high molecular material is heparin modified polyethylene
Or heparin modified polypropylene or heparin modified PVC or heparin modified polyurethane or heparin modified PVA.
The present invention is equipped with annular outer platform 5 in order to hold and handle, in test tube mouth outer wall, and pipe is equipped on test tube mouth
Plug.
The cone cell teeth that tooth array in adsorption structure of the present invention is spaced by altitude range for 0.5-2 millimeters, by that
This spacing range is that the strip groove structural arrangement of 1-3 millimeters deep forms, and wherein ditch piston ring land is equipped with extra projection or groove to increase
Add surface area；Wherein tooth array is formed by sawtooth unit repeated arrangement, and the sawtooth unit is successively arranged 3-6 from top to bottom
The different V-type tooth of length, the length of preferably three V-type teeth are gradually reduced from top to bottom.
Pore diameter range is 0.05-0.5mm in porous structure in adsorption structure of the present invention, hole depth range is
0.05-0.5mm。
The invention also provides a kind of excretion body separation methods using above-mentioned test tube, it is characterised in that including following step
It is rapid:
Step 1: taking above-mentioned test tube, blood to be processed is added in test tube, additional amount is 5-10 milliliters；
Step 2: shaking test tube, come into full contact with sample in test tube with the adsorption structure on inboard wall of test tube；
Step 3: pouring step 2 treated examination liquid in pipe, at this time in sample excretion somatocyst bubble substance can be attached to
On adsorption structure on inboard wall of test tube；
Step 4: tube wall is carefully cleaned multiple times with phosphate buffer
Step 5: appropriate excretion body cracking is added to test tube in step 4 and RNA extracts reagent trizol, or heparin is added
Hydrolase or 8 mol/L sodium chloride solutions dissociation excretion body carry out analysis of protein；
Step 5: being settled by nucleic acid and obtain nucleic acid substances, or pass through antibody binding assay protein ingredient and content.
Embodiment 1:
A kind of test tube for the acquisition of blood excretion body is equipped with test tube main body 1, and inboard wall of test tube, which has, uses heparin modified height
Adsorption structure 2 made of molecular material, the adsorption structure are porous structure, have smooth bottom surface 3, adsorption structure in test tube
2 settings are additionally provided with the groove structure 4 convenient for accelerating the discharge of blood blood cell in test tube lower part, test tube, and the groove structure 4 is set
It sets and is equipped with two or more the grooves 6 around inboard wall of test tube setting in the top of adsorption structure 2, groove structure 4, groove 6 is along test tube
Central axes extend to test tube mouth by the top of adsorption structure；8 V-type ditches that the groove structure 4 is uniformly arranged around inboard wall of test tube
Slot 6, the boss surface between adjacent groove 6 are convexly curved, convenient for accelerating blood or blood cell discharge；The test tube main body
1 is integrally formed using heparin modified high molecular material, or embedding using the embedded type structure made of heparin modified high molecular material
Enter in common test tube, the heparin modified high molecular material is heparin modified PE or heparin modified PVC；In order to hold and handle,
It is equipped with annular outer platform 5 in test tube mouth outer wall, and is equipped with pipe close on test tube mouth；
As shown in Fig. 3, when adsorption structure is porous structure, pore diameter range 0.05-0.5mm, hole depth range are
Blood to be processed is added in test tube when in use by 0.05-0.5mm, and additional amount is 5-10 milliliters；Test tube is shaken, is made in test tube
Sample comes into full contact with the adsorption structure on inboard wall of test tube；Examination liquid in pipe of toppling over that treated, at this time the excretion body in sample
Vesica substance is attached on the adsorption structure on inboard wall of test tube；Tube wall is carefully cleaned multiple times with phosphate buffer；To in step 4
Appropriate excretion body cracking is added in test tube and RNA extracts reagent trizol, and heparin hydrolase or 8 mol/L chlorinations are either added
Sodium solution dissociates excretion body and carries out analysis of protein；It is settled by nucleic acid and obtains nucleic acid substances, or pass through antibody binding assay egg
Bai Chengfen and content.
Embodiment 2:
The present invention proposes a kind of test tube for the acquisition of blood excretion body, is equipped with test tube main body 1, and inboard wall of test tube, which has, to be used
Adsorption structure 2 made of heparin modified high molecular material, the adsorption structure are tooth array, have smooth bottom surface in test tube
3, the groove being additionally provided with as shown in Fig. 2, in test tube in test tube lower part convenient for accelerating the discharge of blood blood cell is arranged in adsorption structure 2
Structure 4, the top that adsorption structure 2 is arranged in of the groove structure 4 are equipped with two or more in groove structure 4 around inboard wall of test tube
The groove 6 of setting, groove 6 extend to test tube mouth by the top of adsorption structure along test tube central axes；The groove structure 4 preferably around
6 or 8 V-grooves 6 that inboard wall of test tube is uniformly arranged, the boss surface between adjacent groove 6 are convexly curved, convenient for plus
Fast blood or blood cell discharge；The test tube main body 1 is integrally formed using heparin modified high molecular material, or using by heparin modified
Embedded type structure made of high molecular material be embedded in common test tube in, the heparin modified high molecular material be heparin modified PE or
Heparin modified PVC；；In order to hold and handle, it is equipped with annular outer platform 5 in test tube mouth outer wall, and pipe close is equipped on test tube mouth.
The cone cell teeth that tooth array in adsorption structure of the present invention is spaced by altitude range for 0.5-2 millimeters, by that
This spacing range is that the strip groove structural arrangement of 1-3 millimeters deep forms, and wherein ditch piston ring land is equipped with extra projection or groove to increase
Add surface area；Wherein tooth array is formed by sawtooth unit repeated arrangement, and the sawtooth unit is successively arranged 3-6 from top to bottom
The different V-type tooth of length, the length of 3-6 V-type tooth are gradually reduced from top to bottom.
As shown in Fig. 3, when adsorption structure is tooth array, tooth array is made of more than two sawtooth units, each
Sawtooth unit includes three V-type teeth being arranged from top to bottom；
When in use, blood to be processed is added in test tube, additional amount is 5-10 milliliters；Test tube is shaken, sample in test tube is made
This comes into full contact with the adsorption structure on inboard wall of test tube；Examination liquid in pipe of toppling over that treated, at this time the excretion somatocyst in sample
Bubble substance is attached on the adsorption structure on inboard wall of test tube；Tube wall is carefully cleaned multiple times with phosphate buffer；To step 4 pilot scale
Appropriate excretion body cracking is added in pipe and RNA extracts reagent Trizol, and heparin hydrolase or 8 mol/L sodium chloride are either added
Solution dissociates excretion body and carries out analysis of protein；It is settled by nucleic acid and obtains nucleic acid substances, or pass through antibody binding assay albumen
Ingredient and content.
Embodiment 3:
The present invention extracts excretion body particle size results in fetal calf serum with ultracentrifugation method and is compared as follows:
As shown in Fig. 5, using excretion body particle diameter distribution obtained by existing ultracentrifugation method as schemed, what this method obtained
Excretion body partial size be (median ± s.d., nm) 71.0 ± 8.1, using as cuvette construction recorded in above-described embodiment 1 it is separated
Excretion body grain size distribution it is as shown in Fig. 6, excretion body partial size be (median ± s.d., nm) 73.4 ± 9.4；Using as above
It is as shown in Fig. 7 to state the separated excretion body grain size distribution of cuvette construction in embodiment 2, is (median ± s.d., nm)
73.8±9.7。
From the above results, excretion body is separated using cuvette construction recorded in the application, can largely guaranteed outer
It secretes somatocyst bubble not to be damaged, disintegrate-quality increases compared with ultracentrifugation method.
The present invention extracts excretion body purity result in fetal calf serum with ultracentrifugation method and is compared as follows:
Wherein excretion bulk concentration calculation method is as follows: concentration standards concentration is denoted as " C1 ", the dilution times of concentration standards
Number scale is " D1 "；Sample to be tested concentration is denoted as " C2 ", and the extension rate of sample to be tested is denoted as " D2 "；Concentration standards detect particle
Number scale is " Q1 ", and product to be tested detection granule number is denoted as " Q2 ", and product to be tested blank control detection granule number is denoted as " Q3 ", then (C1/
D1)/(C2/D2)=Q1/ (Q2-Q3), (C1=2.30x10^11/ml, D1=400, D2=100, Q1=6122, Q3Surpass from=
713, Q3Embodiment 1-2=816).
It is that green is glimmering that attached drawing 8, which is using excretion bulk concentration FITC-SS two dimension scatter plot obtained by existing ultracentrifugation method,
Light-scattering light two dimension scatter plot, gained excretion bulk concentration are 3.78x10^10 (a/ml), and attached drawing 9 is using 1 pilot scale of embodiment
The separated excretion bulk concentration FITC-SS two dimension scatter plot of pipe structure, gained excretion bulk concentration is 4.91x10^10 (a/ml), attached
Figure 10 is using the separated excretion bulk concentration FITC-SS two dimension scatter plot of cuvette construction in embodiment 2, and gained excretion bulk concentration is
(5.15x10^10 a/ml).
From the above results, the separation of excretion body acquired in documented technical solution concentration is not less than now in the application
Some ultracentrifugation methods.
The present invention there is acquisition to facilitate timely compared with the prior art and design, structurally reasonable, easy to operate etc. significant
Advantage.
Claims (10)
1. a kind of test tube for the acquisition of blood excretion body, is equipped with test tube main body, it is characterised in that inboard wall of test tube, which has, uses liver
Specific surface area made of plain modified macromolecule material increases 10 times or more of adsorption structure, and adsorption structure is arranged in test tube lower part.
2. a kind of test tube for the acquisition of blood excretion body according to claim 1, it is characterised in that the adsorption structure
For porous structure or tooth array or FRACTAL ADSORPTION structure or class small intestine wall construction.
3. a kind of test tube for the acquisition of blood excretion body according to claim 1, it is characterised in that be additionally provided in test tube
Convenient for accelerating the groove structure of blood blood cell discharge, the top that adsorption structure is arranged in of the groove structure, in groove structure
Groove equipped with two or more around inboard wall of test tube setting, groove extend to test tube by the top of adsorption structure along test tube central axes
Mouthful.
4. a kind of test tube for the acquisition of blood excretion body according to claim 3, it is characterised in that the groove structure
6-16 V-groove being uniformly arranged around inboard wall of test tube, the boss surface between adjacent groove are convexly curved, convenient for plus
Fast blood or blood cell discharge.
5. a kind of test tube for the acquisition of blood excretion body according to claim 1, it is characterised in that the test tube main body
It is integrally formed using heparin modified high molecular material, or is embedded in using the embedded type structure made of heparin modified high molecular material
In common test tube, the heparin modified high molecular material is heparin modified polyethylene or heparin modified polypropylene or heparin modified
PVC or heparin modified polyurethane or heparin modified PVA.
6. a kind of test tube for the acquisition of blood excretion body according to claim 1, it is characterised in that have light in test tube
Sliding bottom surface.
7. a kind of test tube for the acquisition of blood excretion body according to claim 1, it is characterised in that the adsorption structure
In class small intestine wall construction refer to that inboard wall of test tube is equipped with the policae circulane that specific surface area increases 10 times or more.
8. a kind of test tube for the acquisition of blood excretion body according to claim 2, it is characterised in that the adsorption structure
In tooth array by altitude range be 0.5-2 millimeter interval cone cell teeth, by distance range be 1-3 millimeters deep bar shaped
Groove structure rearranges, and wherein ditch piston ring land is equipped with extra projection or groove to increase surface area；Wherein tooth array is by sawtooth
Unit repeated arrangement is formed, and the sawtooth unit is successively arranged the different V-type tooth of 3-6 length, 3-6 V-type tooth from top to bottom
Length be gradually reduced from top to bottom.
9. a kind of test tube for the acquisition of blood excretion body according to claim 2, it is characterised in that the adsorption structure
In porous structure in pore diameter range be 0.05-0.5mm, hole depth range is 0.05-0.1mm, aperture shape is circular hole or ellipse
Round hole or square hole or elongate holes or tri-angle-holed or pentagon hole or hexagonal hole.
10. a kind of excretion body separation method using the test tube as described in any one of claim 1-9, it is characterised in that including
Following steps:
Step 1: taking above-mentioned test tube, blood to be processed is added in test tube, additional amount is 5-10 milliliters；
Step 2: shaking test tube, come into full contact with sample in test tube with the adsorption structure on inboard wall of test tube；
Step 3: pouring step 2 treated examination liquid in pipe, at this time in sample excretion somatocyst bubble substance attachment in vitro
On adsorption structure on wall；
Step 4: tube wall is carefully cleaned multiple times with phosphate buffer
Step 5: appropriate excretion body cracking is added to test tube in step 4 and RNA extracts reagent trizol, or heparin hydrolysis is added
Enzyme or 8 mol/L sodium chloride solutions dissociation excretion body carry out analysis of protein；
Step 5: being settled by nucleic acid and obtain nucleic acid substances, or pass through antibody binding assay protein ingredient and content.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810963224.0A CN109116010B (en) | 2018-08-22 | 2018-08-22 | Test tube for blood exosome collection and exosome separation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810963224.0A CN109116010B (en) | 2018-08-22 | 2018-08-22 | Test tube for blood exosome collection and exosome separation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109116010A true CN109116010A (en) | 2019-01-01 |
| CN109116010B CN109116010B (en) | 2023-11-21 |
Family
ID=64860112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810963224.0A Active CN109116010B (en) | 2018-08-22 | 2018-08-22 | Test tube for blood exosome collection and exosome separation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109116010B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114199665A (en) * | 2021-12-10 | 2022-03-18 | 谱天（天津）生物科技有限公司 | Method for enriching exosomes in urine |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0727680A (en) * | 1993-07-15 | 1995-01-31 | Hitachi Ltd | Blood corpuscle separating method |
| CN105388055A (en) * | 2015-12-11 | 2016-03-09 | 浙江省肿瘤医院 | Method for separating tumor cell derived-exosomes from urine |
| CN105934670A (en) * | 2013-12-03 | 2016-09-07 | 拜奥默里克斯公司 | Method for isolating exosomes |
| 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 |
| CN106289926A (en) * | 2016-07-26 | 2017-01-04 | 华东理工大学 | A kind of method using immuno magnetic cell separation serum China and foreign countries to secrete body |
| CN107254430A (en) * | 2017-08-11 | 2017-10-17 | 上海浦美生物医药科技有限公司 | A kind of method based on positive charge adsorbing separation excretion body |
| CN107893051A (en) * | 2017-10-11 | 2018-04-10 | 北京大学 | A kind of method of excretion body in serum using immuno magnetic cell separation |
| CN208888247U (en) * | 2018-08-22 | 2019-05-21 | 威海纽兰生物科技有限公司 | Test tube for the acquisition of blood excretion body |
-
2018
- 2018-08-22 CN CN201810963224.0A patent/CN109116010B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0727680A (en) * | 1993-07-15 | 1995-01-31 | Hitachi Ltd | Blood corpuscle separating method |
| CN105934670A (en) * | 2013-12-03 | 2016-09-07 | 拜奥默里克斯公司 | Method for isolating exosomes |
| CN105388055A (en) * | 2015-12-11 | 2016-03-09 | 浙江省肿瘤医院 | Method for separating tumor cell derived-exosomes from urine |
| 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 |
| CN106289926A (en) * | 2016-07-26 | 2017-01-04 | 华东理工大学 | A kind of method using immuno magnetic cell separation serum China and foreign countries to secrete body |
| CN107254430A (en) * | 2017-08-11 | 2017-10-17 | 上海浦美生物医药科技有限公司 | A kind of method based on positive charge adsorbing separation excretion body |
| CN107893051A (en) * | 2017-10-11 | 2018-04-10 | 北京大学 | A kind of method of excretion body in serum using immuno magnetic cell separation |
| CN208888247U (en) * | 2018-08-22 | 2019-05-21 | 威海纽兰生物科技有限公司 | Test tube for the acquisition of blood excretion body |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114199665A (en) * | 2021-12-10 | 2022-03-18 | 谱天（天津）生物科技有限公司 | Method for enriching exosomes in urine |
| CN114199665B (en) * | 2021-12-10 | 2024-02-09 | 谱天（天津）生物科技有限公司 | Enrichment method of exosomes in urine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109116010B (en) | 2023-11-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8900843B2 (en) | Kit and method for the capture of tumor cells | |
| CN106399250A (en) | Method and kit for separating exosome | |
| RU2503009C2 (en) | Apparatus and method for blood separation and analysis | |
| US20110195413A1 (en) | Integrated Method for Enriching and Detecting Rare Cells from Biological Body Fluid Sample | |
| ES2546837T3 (en) | Target cell isolation procedure | |
| US20180299425A1 (en) | Methods and Apparatus for Segregation of Particles | |
| CN108795869A (en) | A kind of circulating tumor cell positive enrichment method | |
| RU99110373A (en) | USE OF ANTI-BODIES AGAINST EMBRYONAL HEMOGLOBIN FOR IDENTIFICATION OF FETAL CELLS | |
| CN85104030A (en) | Method of immunity and device | |
| ITTO20060833A1 (en) | MICROPOZZETTO CONVEXED IN ORDER TO CREATE A PERIMETRAL ROOM FOR THE COLLECTION OF CORPUSCULATED ELEMENTS | |
| CA2782176A1 (en) | Methods and apparatus for segregation of particles, including segregation and proliferation of fetal and stem cells | |
| CN208888247U (en) | Test tube for the acquisition of blood excretion body | |
| WO2021110939A1 (en) | Methods for identifying viral infections and for analyzing exosomes in liquid samples by raman spectroscopy | |
| CN105242036B (en) | A kind of electrochemiluminescent immunoassay detection integrated Reagent Tube and using method thereof | |
| CN107076740A (en) | The detection method of targeting molecule and kit wherein used | |
| CN109116010A (en) | Test tube and excretion body separation method for the acquisition of blood excretion body | |
| CN109913417A (en) | A method of using different cell origin excretion body hypotypes in tachysynthesis paramagnetic particle method separation cerebrospinal fluid | |
| CN110174519B (en) | Confluent-detection type erythrocyte blood type irregular antibody detection kit based on solid-phase agglutination technology and preparation method thereof | |
| CN105842464B (en) | Joint based on up-converting phosphor technology quantitatively detects uNGAL and uCr device and preparation method thereof | |
| US20220241775A1 (en) | Sampling device for biological specimen | |
| US9632086B2 (en) | Method and kit for determining-antibody sensitivity and clone cell strain | |
| CN206095941U (en) | Specific marker circulating tumor cell immunoprecipitation reaction detection box | |
| CN108660060B (en) | Microfluidic chip for enriching and purifying circulating tumor cells | |
| CN105759029A (en) | Detection kit for measles virus and application method of detection kit | |
| CN109827806A (en) | A kind of acquisition device and method of circulating tumor cell |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |