CN109116010B - Test tube for blood exosome collection and exosome separation method - Google Patents
Test tube for blood exosome collection and exosome separation method Download PDFInfo
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- 238000012360 testing method Methods 0.000 title claims abstract description 133
- 210000001808 exosome Anatomy 0.000 title claims abstract description 79
- 210000004369 blood Anatomy 0.000 title claims abstract description 33
- 239000008280 blood Substances 0.000 title claims abstract description 33
- 238000000926 separation method Methods 0.000 title abstract description 10
- 238000001179 sorption measurement Methods 0.000 claims abstract description 57
- 239000002861 polymer material Substances 0.000 claims abstract description 20
- 229960002897 heparin Drugs 0.000 claims abstract description 10
- 229920000669 heparin Polymers 0.000 claims abstract description 10
- 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 5
- 238000000034 method Methods 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 210000000601 blood cell Anatomy 0.000 claims description 10
- 239000011148 porous material Substances 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- -1 polyethylene Polymers 0.000 claims description 6
- 239000004698 Polyethylene Substances 0.000 claims description 5
- 238000002123 RNA extraction Methods 0.000 claims description 5
- 238000003916 acid precipitation Methods 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 5
- 238000003776 cleavage reaction Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 230000007017 scission Effects 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 239000004743 Polypropylene Substances 0.000 claims description 3
- 238000004140 cleaning Methods 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
- 230000001133 acceleration Effects 0.000 claims 1
- 239000002245 particle Substances 0.000 description 11
- 238000000703 high-speed centrifugation Methods 0.000 description 8
- 238000010586 diagram Methods 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 3
- 210000000813 small intestine Anatomy 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 241000973497 Siphonognathus argyrophanes Species 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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- 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
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- 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
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- 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
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- Hematology (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The application relates to the technical field of molecular biology and clinical examination, in particular to a test tube for collecting blood exosomes and a separation method of exosomes, which are reasonable in structure and simple and convenient to operate, and are characterized in that the inner wall of the test tube is provided with an adsorption structure which is made of heparin modified high polymer materials and has the specific surface area increased by more than 10 times, the adsorption structure is arranged at the lower part of the test tube, and the adsorption structure is a porous structure, a sawtooth array, a fractal array or a small intestine-like wall structure.
Description
Technical Field
The application relates to the technical field of molecular biology and clinical examination, in particular to a test tube for collecting blood exosomes and an exosome separation method, which have reasonable structure and simple operation and do not need centrifugation to remove blood cells in a sample.
Background
Exosomes are small vesicles 30-150nm in diameter, selectively packaged and released by living cells. The content of the extract in body fluids of human bodies, such as blood, urine, cerebrospinal fluid and the like, is rich. The exosomes contain different kinds of lipids, nucleic acids, proteins, etc., which can be transported to specific target cells, thereby exerting corresponding biological functions. Thus, exosomes play a tremendous role in intercellular communication and in physiological and pathological processes.
At present, three methods for extracting serum exosomes are most commonly used, namely, an ultra-high speed centrifugation method, an immunomagnetic bead method and a kit method, such as related serum exosomes preparation reagents provided by Thermo company and SBI company in the United states. However, all three have obvious defects, the purity of the obtained exosomes is high by an ultra-high speed centrifugation method, but the yield is low, the time consumption is long, the operation is complex, the instrument is expensive and is not suitable for the detection practice of a hospital, and the repeated centrifugation operation can possibly damage vesicles, so that the quality of the vesicles is reduced; the immunomagnetic bead method can specifically capture corresponding exosomes, but the technology has high antibody cost, which is mastered by a few international huge companies, such as the American SBI company, and the like, and has high cost; the yield of extracting exosomes by a sedimentation kit method is high, but the purity of the exosomes is too low, and a plurality of proteins with high abundance in serum can be obtained while the exosomes are obtained. In addition, existing exosome separation techniques are usually validated by Western blot after a period of infection and cannot be monitored in real time.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the application provides a test tube for collecting blood exosomes and an exosome separation method, which are free of centrifugal treatment, reasonable in structure and simple and convenient to operate.
The application can be achieved by the following measures:
a test tube for collecting blood exosome is provided with a test tube main body, and is characterized in that the inner wall of the test tube is provided with an adsorption structure which is made of heparin modified polymer material and has the specific surface area increased by more than 10 times, and the adsorption structure is arranged at the lower part of the test tube.
The adsorption structure is a porous structure, a sawtooth array or a fractal array adsorption structure or a small intestine wall-like structure.
The application also discloses a groove structure which is convenient for accelerating the discharge of blood and blood cells, the groove structure is arranged above the adsorption structure, more than two grooves which are arranged around the inner wall of the test tube are arranged in the groove structure, and the grooves extend to the test tube port from the top end of the adsorption structure along the central axis of the test tube.
The groove structure of the application is uniformly provided with 6-16V-shaped grooves around the inner wall of the test tube, and the boss surfaces between adjacent grooves are in an outwards convex arc shape, so that the discharge of blood or blood cells is accelerated.
The main body of the test tube is integrally formed by adopting a heparin-modified polymer material, or is embedded into a common test tube by adopting an embedded structure made of the heparin-modified polymer material, wherein the heparin-modified polymer material is heparin-modified polyethylene or heparin-modified polypropylene or heparin-modified PVC or heparin-modified polyurethane or heparin-modified PVA.
The application has smooth bottom surface in the test tube, which is convenient for discharging blood.
For holding and processing, the outer wall of the test tube port is provided with an annular outer boss, and the test tube port is provided with a tube plug.
The small intestine wall-like structure in the adsorption structure of the application means that the inner wall of the test tube is provided with annular folds with the specific surface area increased by more than 10 times, and the folds are folds.
The sawtooth array in the adsorption structure is formed by arraying conical teeth with the height range of 0.5-2 mm at intervals according to a strip-shaped groove structure with the distance range of 1-3 mm, wherein the groove lands are provided with additional bulges or grooves to increase the surface area, the sawtooth array is formed by repeatedly arraying sawtooth units, 3-6V-shaped teeth with different lengths are sequentially arranged on the sawtooth units from top to bottom, and the lengths of the 3-6V-shaped teeth are gradually reduced from top to bottom.
The porous structure in the adsorption structure has the pore diameter range of 0.05-0.5mm and the pore depth range of 0.05-0.1mm, and the pore diameter is round holes or elliptical holes or square holes or strip holes or triangular holes or pentagonal holes or hexagonal holes.
The application also provides an exosome separation method by using the test tube, which is characterized by comprising the following steps:
step 1: taking the test tube, and adding the blood to be treated into the test tube with the addition amount of 5-10 milliliters;
step 2: shaking the test tube to make the sample in the test tube fully contact with the adsorption structure on the inner wall of the test tube;
step 3: pouring the liquid in the test tube treated in the step 2, wherein exosome vesicle substances in the sample are attached to an adsorption structure on the inner wall of the test tube;
step 4, carefully cleaning the pipe wall for many times by using phosphate buffer solution
Step 5: adding a proper amount of exosome cleavage and RNA extraction reagent trizol into the test tube in the step 4, or adding heparin hydrolase or 8 mol/L sodium chloride solution to dissociate exosome for protein analysis;
step 5: nucleic acid material is obtained by nucleic acid precipitation or protein composition and content are analyzed by antibody binding.
Compared with the prior art and design, the application has the remarkable advantages of convenient and timely collection, reasonable structure, simple and convenient operation and the like.
Drawings
Fig. 1 is a schematic diagram of the structure of the present application.
Fig. 2 is a cross-sectional view of the trench structure of fig. 1.
Fig. 3 is a schematic diagram of an adsorption structure in the present application, wherein fig. 3-1 is a first structural schematic diagram of the adsorption structure, fig. 3-2 is a second structural schematic diagram of the adsorption structure, and fig. 3-3 is a third structural schematic diagram of the adsorption structure.
Fig. 4 is a schematic structural view of an embodiment of the saw tooth array according to the present application.
FIG. 5 is a graph showing the particle size distribution of exosomes obtained by separating exosomes by conventional centrifugation in example 3.
FIG. 6 is a graph showing the particle size distribution of exosomes obtained in example 3 using the test tube configuration of example 1.
FIG. 7 is a graph showing the particle size distribution of exosomes obtained in example 3 using the pilot tube configuration of example 2.
FIG. 8 is a two-dimensional scatter plot of the exosome concentration FITC-SS obtained in example 3 using conventional centrifugation to isolate the exosome.
FIG. 9 is a two-dimensional scatter plot of the exosome concentration FITC-SS obtained in example 3 using the pilot tube configuration of example 1 to isolate exosomes.
FIG. 10 is a two-dimensional scatter plot of exosome concentration FITC-SS obtained in example 3 using the pilot tube configuration of example 2 to isolate exosomes.
Reference numerals: test tube main part 1, adsorption structure 2, bottom surface 3, slot structure 4, annular outer boss 5, slot 6.
Detailed Description
The application is further described below with reference to the drawings and examples.
The application provides a test tube for collecting blood exosomes, which is provided with a test tube main body and is characterized in that the inner wall of the test tube is provided with an adsorption structure which is made of heparin modified polymer materials and has a specific surface area increased by more than 10 times, the adsorption structure is a porous structure or a sawtooth array or a fractal adsorption structure or a small intestine wall structure, a smooth bottom surface 3 is arranged in the test tube, the adsorption structure 2 is arranged at the lower part of the test tube, a groove structure 4 which is convenient for accelerating blood discharge is also arranged in the test tube, the groove structure 4 is arranged above the adsorption structure 2, more than two grooves 6 which are arranged around the inner wall of the test tube are arranged in the groove structure 4, and the grooves 6 extend from the top end of the adsorption structure to a test tube port along the central axis of the test tube.
The groove structure 4 is preferably provided with 6 or 8V-shaped grooves 6 uniformly arranged around the inner wall of the test tube, and the surface of a boss between every two adjacent grooves 6 is in an outwards convex arc shape, so that the discharge of blood or blood cells is accelerated.
The test tube main body 1 is integrally formed by adopting a heparin-modified polymer material, or is embedded into a common test tube by adopting an embedded structure made of the heparin-modified polymer material, wherein the heparin-modified polymer material is heparin-modified polyethylene or heparin-modified polypropylene or heparin-modified PVC or heparin-modified polyurethane or heparin-modified PVA.
For holding and processing, the outer wall of the test tube port is provided with an annular outer boss 5, and the test tube port is provided with a tube plug.
The sawtooth array in the adsorption structure is formed by arraying conical teeth with the height range of 0.5-2 mm at intervals according to a strip-shaped groove structure with the distance range of 1-3 mm, wherein the groove land is provided with additional protrusions or grooves to increase the surface area, the sawtooth array is formed by repeatedly arraying sawtooth units, 3-6V-shaped teeth with different lengths are sequentially arranged on the sawtooth units from top to bottom, and the lengths of three V-shaped teeth are preferably gradually reduced from top to bottom.
The pore diameter range in the porous structure in the adsorption structure is 0.05-0.5mm, and the pore depth range is 0.05-0.5mm.
The application also provides an exosome separation method by using the test tube, which is characterized by comprising the following steps:
step 1: taking the test tube, and adding the blood to be treated into the test tube with the addition amount of 5-10 milliliters;
step 2: shaking the test tube to make the sample in the test tube fully contact with the adsorption structure on the inner wall of the test tube;
step 3: pouring the liquid in the test tube treated in the step 2, wherein exosome vesicle substances in the sample are attached to an adsorption structure on the inner wall of the test tube;
step 4, carefully cleaning the pipe wall for many times by using phosphate buffer solution
Step 5: adding a proper amount of exosome cleavage and RNA extraction reagent trizol into the test tube in the step 4, or adding heparin hydrolase or 8 mol/L sodium chloride solution to dissociate exosome for protein analysis;
step 5: nucleic acid material is obtained by nucleic acid precipitation or protein composition and content are analyzed by antibody binding.
Examples
The utility model provides a test tube for blood exosome collection, is equipped with test tube main part 1, and the test tube inner wall has the adsorption structure 2 that adopts heparin to decorate macromolecular material and make, adsorption structure is porous structure, has smooth bottom surface 3 in the test tube, and adsorption structure 2 sets up in the test tube lower part, still is equipped with the slot structure 4 that is convenient for accelerate blood corpuscle emission in the test tube, the setting of slot structure 4 is in the top of adsorption structure 2, is equipped with more than two slots 6 that set up around the test tube inner wall in the slot structure 4, and slot 6 extends to the test tube mouth from the top of adsorption structure along the test tube axis; the groove structure 4 is uniformly provided with 8V-shaped grooves 6 around the inner wall of the test tube, and the boss surfaces between the adjacent grooves 6 are in an outwards convex arc shape, so that the discharge of blood or blood cells is accelerated; the test tube main body 1 is integrally formed by adopting a heparin-modified polymer material, or is embedded into a common test tube by adopting an embedded structure made of the heparin-modified polymer material, wherein the heparin-modified polymer material is heparin-modified PE or heparin-modified PVC; for holding and processing, an annular outer boss 5 is arranged on the outer wall of the test tube port, and a tube plug is arranged on the test tube port;
as shown in figure 3, when the adsorption structure is a porous structure, the pore diameter range is 0.05-0.5mm, the pore depth range is 0.05-0.5mm, and when in use, the blood to be treated is added into a test tube, and the addition amount is 5-10 milliliters; shaking the test tube to make the sample in the test tube fully contact with the adsorption structure on the inner wall of the test tube; pouring the treated liquid in the test tube, wherein exosome vesicle substances in the sample are attached to an adsorption structure on the inner wall of the test tube; carefully washing the tube wall with phosphate buffer solution for many times; adding a proper amount of exosome cleavage and RNA extraction reagent trizol into the test tube in the step 4, or adding heparin hydrolase or 8 mol/L sodium chloride solution to dissociate exosome for protein analysis; nucleic acid material is obtained by nucleic acid precipitation or protein composition and content are analyzed by antibody binding.
Examples
The application provides a test tube for collecting blood exosomes, which is provided with a test tube main body 1, wherein the inner wall of the test tube is provided with an adsorption structure 2 made of heparin-modified polymer materials, the adsorption structure is a saw-tooth array, a smooth bottom surface 3 is arranged in the test tube, the adsorption structure 2 is arranged at the lower part of the test tube, as shown in figure 2, the test tube is also provided with a groove structure 4 which is convenient for accelerating the discharge of blood corpuscles, the groove structure 4 is arranged above the adsorption structure 2, more than two grooves 6 which are arranged around the inner wall of the test tube are arranged in the groove structure 4, and the grooves 6 extend from the top end of the adsorption structure to a test tube port along the central axis of the test tube; the groove structure 4 is preferably provided with 6 or 8V-shaped grooves 6 uniformly arranged around the inner wall of the test tube, and the surface of a boss between every two adjacent grooves 6 is in an outwards convex arc shape, so that the discharge of blood or blood cells is accelerated; the test tube main body 1 is integrally formed by adopting a heparin-modified polymer material, or is embedded into a common test tube by adopting an embedded structure made of the heparin-modified polymer material, wherein the heparin-modified polymer material is heparin-modified PE or heparin-modified PVC; the method comprises the steps of carrying out a first treatment on the surface of the For holding and processing, an annular outer boss 5 is arranged on the outer wall of the test tube port, and a tube plug is arranged on the test tube port.
The sawtooth array in the adsorption structure is formed by arraying conical teeth with the height range of 0.5-2 mm at intervals according to a strip-shaped groove structure with the distance range of 1-3 mm, wherein the groove lands are provided with additional bulges or grooves to increase the surface area, the sawtooth array is formed by repeatedly arraying sawtooth units, 3-6V-shaped teeth with different lengths are sequentially arranged on the sawtooth units from top to bottom, and the lengths of the 3-6V-shaped teeth are gradually reduced from top to bottom.
As shown in fig. 3, when the adsorption structure is a sawtooth array, the sawtooth array is composed of more than two sawtooth units, and each sawtooth unit comprises three V-shaped teeth arranged from top to bottom;
when in use, the blood to be treated is added into a test tube with the addition amount of 5-10 milliliters; shaking the test tube to make the sample in the test tube fully contact with the adsorption structure on the inner wall of the test tube; pouring the treated liquid in the test tube, wherein exosome vesicle substances in the sample are attached to an adsorption structure on the inner wall of the test tube; carefully washing the tube wall with phosphate buffer solution for many times; adding a proper amount of exosome cleavage and RNA extraction reagent Trizol into the test tube in the step 4, or adding heparin hydrolase or 8 mol/L sodium chloride solution to dissociate exosome for protein analysis; nucleic acid material is obtained by nucleic acid precipitation or protein composition and content are analyzed by antibody binding.
Examples
Compared with the result of extracting the grain size of exosomes in the fetal bovine serum by using an ultra-high speed centrifugation method, the method provided by the application has the following steps:
as shown in FIG. 5, the particle size distribution of the exosomes obtained by the conventional ultra-high speed centrifugation method is shown in the graph, the particle size of the exosomes obtained by the method is (media + -s.d., nm) 71.0 + -8.1, and the particle size distribution of the exosomes separated by the test tube structure as described in the above example 1 is shown in FIG. 6, the particle size of the exosomes is (media + -s.d., nm) 73.4 + -9.4; the exosome particle size distribution profile isolated using the pilot tube structure described in example 2 above is (media±s.d., nm) 73.8±9.7 as shown in fig. 7.
From the above results, it is clear that the exosome can be separated by using the test tube structure described in the present application, so that the exosome vesicles can be prevented from being damaged to a large extent, and the separation quality can be improved compared with that of the ultra-high speed centrifugation method.
Compared with the result of extracting exosome purity in the fetal bovine serum by using an ultra-high speed centrifugation method, the method provided by the application has the following steps:
wherein the exosome concentration calculation method is as follows: the concentration of the concentration standard substance is marked as C1, and the dilution number of the concentration standard substance is marked as D1; the concentration of the sample to be measured is marked as C2, and the dilution number of the sample to be measured is marked as D2; the concentration standard substance detection particle number is marked as "Q1", the test substance detection particle number is marked as "Q2", and the test substance blank control detection particle number is marked as "Q3", then (C1/D1)/(C2/D2) =Q1/(Q2-Q3), (C1=2.30x10ζ11 pieces/ml, d1=400, d2=100, Q1=6122, Q3) Super-release =713，Q3 Examples 1 to 2 =816）。
FIG. 8 is a green fluorescence-scattered light two-dimensional scattergram, which is a FITC-SS two-dimensional scattergram of the exosome concentration obtained by the conventional ultra-high speed centrifugation, and the exosome concentration obtained was 3.78x10ζ10 (in/ml), FIG. 9 is a FITC-SS two-dimensional scattergram of the exosome concentration separated by the test tube structure of example 1, and the exosome concentration obtained was 4.91x10ζ10 (in/ml), and FIG. 10 is a FITC-SS two-dimensional scattergram of the exosome concentration separated by the test tube structure of example 2, and the exosome concentration obtained was 5.15x10ζ10 (in/ml).
From the above results, it is clear that the exosome separation concentration obtained by the technical scheme described in the present application is not lower than that obtained by the conventional ultra-high-speed centrifugation method.
Compared with the prior art and design, the application has the remarkable advantages of convenient and timely collection, reasonable structure, simple and convenient operation and the like.
Claims (10)
1. A test tube for collecting blood exosome is provided with a test tube main body, and is characterized in that the inner wall of the test tube is provided with an adsorption structure which is made of heparin modified polymer material and has the specific surface area increased by more than 10 times, and the adsorption structure is arranged at the lower part of the test tube.
2. A test tube for blood exosome collection according to claim 1, characterized in that the adsorption structure is a porous structure or a zigzag array or a fractal adsorption structure or a small intestine-like wall structure.
3. The test tube for blood exosome collection according to claim 1, wherein the test tube is further provided with a groove structure which is convenient for accelerating blood and blood cell discharge, the groove structure is arranged above the adsorption structure, more than two grooves which are arranged around the inner wall of the test tube are arranged in the groove structure, and the grooves extend from the top end of the adsorption structure to the test tube port along the central axis of the test tube.
4. A test tube for blood exosome collection according to claim 3, wherein the groove structure is 6-16V-shaped grooves uniformly arranged around the inner wall of the test tube, and the boss surface between adjacent grooves is in an outwardly convex arc shape, so as to facilitate the acceleration of the discharge of blood or blood cells.
5. The test tube for blood exosome collection according to claim 1, wherein the test tube body is integrally formed by heparin-modified polymer material, or is embedded in a common test tube by an embedded structure made of heparin-modified polymer material, and the heparin-modified polymer material is heparin-modified polyethylene or heparin-modified polypropylene or heparin-modified PVC or heparin-modified polyurethane or heparin-modified PVA.
6. A test tube for blood exosome collection according to claim 1, wherein the test tube has a smooth bottom surface.
7. The test tube for blood exosome collection according to claim 1, wherein the small intestine-like wall structure in the adsorption structure means that annular folds with a specific surface area increased by more than 10 times are arranged on the inner wall of the test tube.
8. A test tube for blood exosome collection according to claim 2, wherein the array of serrations in the adsorption structure consists of conical teeth with a height ranging from 0.5 to 2 mm apart, arranged in a stripe-shaped groove structure with a depth ranging from 1 to 3 mm apart from each other, wherein the groove lands are provided with additional protrusions or grooves to increase the surface area; the sawtooth array is formed by repeatedly arranging sawtooth units, 3-6V-shaped teeth with different lengths are sequentially arranged on the sawtooth units from top to bottom, and the lengths of the 3-6V-shaped teeth are gradually reduced from top to bottom.
9. A test tube for blood exosome collection according to claim 2, characterized in that the porous structure in the adsorption structure has a pore size in the range of 0.05-0.5mm and a pore depth in the range of 0.05-0.1mm, and the pore size is in the shape of a round hole or an oval hole or a square hole or a rectangular hole or a triangular hole or a pentagonal hole or a hexagonal hole.
10. A method for separating exosomes using the test tube of any one of claims 1-9, characterized by comprising the steps of:
step 1: taking the test tube, and adding the blood to be treated into the test tube with the addition amount of 5-10 milliliters;
step 2: shaking the test tube to make the sample in the test tube fully contact with the adsorption structure on the inner wall of the test tube;
step 3: pouring the liquid in the test tube treated in the step 2, wherein exosome vesicle substances in the sample are attached to an adsorption structure on the inner wall of the test tube;
step 4, carefully cleaning the pipe wall for many times by using phosphate buffer solution;
step 5: adding a proper amount of exosome cleavage and RNA extraction reagent trizol into the test tube in the step 4, or adding heparin hydrolase or 8 mol/L sodium chloride solution to dissociate exosome for protein analysis;
step 5: nucleic acid material is obtained by nucleic acid precipitation or protein composition and content are analyzed by antibody binding.
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 |
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| 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 |
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| CN109116010A CN109116010A (en) | 2019-01-01 |
| CN109116010B true CN109116010B (en) | 2023-11-21 |
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