CN110075942B - Biological fluid sampling and separating device and method capable of realizing solid-liquid phase isolation after centrifugation - Google Patents
Biological fluid sampling and separating device and method capable of realizing solid-liquid phase isolation after centrifugation Download PDFInfo
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- CN110075942B CN110075942B CN201910381724.8A CN201910381724A CN110075942B CN 110075942 B CN110075942 B CN 110075942B CN 201910381724 A CN201910381724 A CN 201910381724A CN 110075942 B CN110075942 B CN 110075942B
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- 239000007791 liquid phase Substances 0.000 title claims abstract description 59
- 238000005119 centrifugation Methods 0.000 title claims abstract description 41
- 239000013060 biological fluid Substances 0.000 title claims abstract description 27
- 238000002955 isolation Methods 0.000 title claims abstract description 22
- 238000005070 sampling Methods 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000012360 testing method Methods 0.000 claims abstract description 156
- 239000007790 solid phase Substances 0.000 claims abstract description 22
- 239000004033 plastic Substances 0.000 claims description 28
- 229920003023 plastic Polymers 0.000 claims description 28
- 230000009471 action Effects 0.000 claims description 23
- 238000000926 separation method Methods 0.000 claims description 18
- 238000005191 phase separation Methods 0.000 claims description 10
- 238000001746 injection moulding Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 2
- 238000010276 construction Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 4
- 239000002245 particle Substances 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 20
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 238000000605 extraction Methods 0.000 description 8
- 230000004224 protection Effects 0.000 description 7
- 210000000601 blood cell Anatomy 0.000 description 5
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 206010048612 Hydrothorax Diseases 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000032798 delamination Effects 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000028937 DNA protection Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
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- 239000002184 metal Substances 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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Classifications
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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
-
- 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
- B01L3/50825—Closing or opening means, corks, bungs
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Centrifugal Separators (AREA)
Abstract
The invention discloses a biological fluid sampling and separating device capable of realizing solid-liquid phase isolation after centrifugation, which comprises a test tube, a guide rail shell, a thimble ring, a clamp ring, a rotary tooth column, a thrust tooth column, a push rod and a spring, wherein the outer diameter of the test tube is of a thick structure at two thin ends, the guide rail shell is of a cylinder structure with a cavity, and the thimble ring, the clamp ring, the rotary tooth column and the thrust tooth column are sequentially sleeved on the test tube and are placed in the inner cavity of the guide rail shell. By using the device, the physical isolation between the solid phase and the liquid phase layer can be automatically realized after the solid-liquid phase layering of the biological fluid sample containing solid phase particle components is completed through centrifugation, so that target detection substances such as free DNA in the liquid phase layer are protected from being polluted by solid phase substances such as cells in the sample preservation and transportation processes.
Description
Technical Field
The invention relates to a biological fluid sampling and separating device and method capable of realizing solid-liquid phase separation after centrifugation, and belongs to the field of sample collection.
Background
Free DNA (cfDNA) is an extracellular free DNA fragment that is present in various body fluids including blood, urine, hydrothorax, milk, etc. Peripheral blood cfDNA has been used to perform noninvasive prenatal gene testing (NIPT); peripheral blood and hydrothorax cfDNA can be used for concomitant diagnosis of tumor targeting drugs; early screening of cfDNA-based cancers is also of great interest and research. When cfDNA is used for various assays, it is an important prerequisite to protect cfDNA from degradation and contamination by blood cell genomic DNA during body fluid collection, preservation and transport. The only protection method at present is to add an anticoagulant and DNA protection mixed reagent into a vacuum blood collection tube, and the vacuum blood collection tube can be preserved for 1-2 weeks at the temperature of 4-30 ℃, but the protection effect is not ideal at the temperature higher than 35 ℃, and particularly the preservation and transportation at the temperature below 0 ℃ are completely impossible, because the high temperature and the freezing and thawing can cause the cells to release the genome DNA. Therefore, samples such as blood for cfDNA detection are difficult to transport and store safely in summer and winter, have high cost, and the reliability of cfDNA detection results is reduced due to factors of environment and severe vibration.
The fundamental method for solving the problem is that the collected sample is subjected to solid-liquid phase separation (e.g. manual operation is performed to separate plasma and blood cells into different containers after centrifugation) and then the sample is transported. However, the current sample collection method requires professional experimenters to operate the sample collection method to realize manual separation before transportation, and has great risks of misoperation.
Disclosure of Invention
The technical solution of the invention is as follows: in order to overcome the defects of the prior art, the device and the method for sampling and separating the biological fluid, which can realize solid-liquid phase separation after centrifugation, are provided, the device ingeniously utilizes the elasticity of a spring and the centrifugal force, and after the solid-liquid phase centrifugation and layering of a sample are finished, the solid-liquid phase physical separation is automatically realized, the manual separation operation is not needed, and powerful guarantee is provided for the stable transportation of the sample containing cfDNA under the extreme temperature condition and the whole-course automation of the follow-up cfDNA extraction.
The technical solution of the invention is as follows:
the biological fluid sampling and separating device capable of realizing solid-liquid phase isolation after centrifugation comprises a test tube, a guide rail shell, a thimble ring, a clamp ring, a rotary tooth column, a thrust tooth column, a push rod and a spring,
the test tube is of a thick structure at two thin ends in the middle, one end of an opening of the test tube is sleeved on the small diameter of the inner cavity of the test tube connecting ring, the spring is arranged in a cavity formed by the outer wall of the test tube and the test tube connecting ring, one end of the guide rail shell is fixedly connected with the large diameter end of the inner cavity of the test tube connecting ring, and one end of the push rod can enter from the other end of the guide rail shell;
the guide rail shell is of a cylindrical structure with a cavity, clamping grooves are uniformly arranged on the inner wall of the guide rail shell, which is close to one end of the test tube connecting ring, along the axial circumferential direction, and protruding guide rails are uniformly arranged on the inner wall of the guide rail shell, which is close to one end of the push rod, along the axial circumferential direction;
the thimble ring, the clamp ring, the rotary tooth column and the thrust tooth column are sleeved on the test tube in sequence and are arranged in the inner cavity of the guide rail shell;
the thimble ring is of a hollow cavity structure, the thimble which is symmetrically designed is fixedly connected with one end of the thimble ring which is close to one end of the test tube connecting ring along the axial circumferential direction,
the clamp ring is coaxially sleeved on the thimble ring, the clamp ring is fixedly connected with the guide rail shell, and the thimble ring and the clamp ring can slide relatively along the circumferential direction; the clamp ring is axially provided with a thimble groove matched with the thimble, the inner wall of the clamp ring is symmetrically connected with a C-shaped clamp, and the initial state of the C-shaped clamp is in an open ring shape;
the rotary tooth column is of a hollow column structure, the outer wall of the rotary tooth column is uniformly provided with rotary tooth column grooves along the axial circumferential direction, one end of the rotary tooth column is coaxially contacted with the other end of the thimble ring,
the thrust tooth post is cavity cylindricality structure, evenly opens along axial circumference on its outer wall has thrust tooth post recess, and thrust tooth post recess is the cooperation with the protruding guide rail of guide rail shell all the time, and thrust tooth post one end is the dislocation meshing with rotatory tooth post other end, and thrust tooth post other end and push rod one end contact.
When in operation, the device comprises: the device is placed into a centrifugal machine for centrifugation after a biological fluid sample is collected, a solid phase and a liquid phase are separated under the action of centrifugal force, meanwhile, under the action of the centrifugal force, a thrust tooth column moves axially along a convex guide rail of a guide rail shell, the acting force of a spring is overcome, the thrust tooth column drives a rotary tooth column and a thimble ring to move synchronously, when the tooth tip of the rotary tooth column exceeds the convex guide rail, the rotary tooth column rotates under the action of the spring force and guided by an inclined plane at the top end of the tooth column, and at the moment, a C-shaped clamp is in an open state; when the centrifugation is finished, the rotation speed is reduced, the centrifugal force is gradually reduced, the spring force is gradually larger than the centrifugal force, the spring force pushes the thimble ring, the thimble ring pushes the rotary tooth column to slide into the convex guide rail and move reversely along the convex guide rail, the thimble ring, the rotary tooth column and the thrust tooth column move reversely together, the thimble of the thimble ring is arranged in the thimble groove of the clamp ring, and meanwhile the thimble acts on the C-shaped clamp of the clamp ring, so that the closing of the two C-shaped clamps is realized, the liquid flow channel of the test tube is closed, and the automatic solid-liquid phase physical isolation is realized.
The distance between the thimble in the initial state and the C-shaped clamp is smaller than the distance of the backward movement of the subsequent rotary tooth column.
The test tube is the combination of different hardness plastics material, includes any one of following schemes:
scheme 1: the range from 5-15mm upwards of the C-shaped clamp to 5-15mm downwards of the C-shaped clamp is soft plastic, and the Shore hardness A is 50-70; the other parts of the test tube are made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring and the test tube are an injection molding whole;
scheme 2: the range from the bottom of the test tube to the upward 5-15mm of the C-shaped clamp is soft plastic, and the Shore A hardness is 50-70; the other parts of the test tube are made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring and the test tube are an injection molding whole;
scheme 3: the inner core of the test tube is made of soft plastic, the Shore hardness A is 50-70, the test tube connecting ring sleeved at one end of the opening of the test tube is made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring and the test tube are tightly clamped and glued into a whole through the outer wall structure at one end of the opening of the test tube.
The whole length of the test tube is 9-13mm, the inner diameter of the test tube is 7-11mm, the distance from the bottom of the test tube to the top end of the closed C-shaped clamp is 3-8mm, and the wall thickness of the test tube is 0.5-2.5mm.
Preferably, the whole length of the test tube is 11mm, the inner diameter of the test tube is 9mm, the distance from the bottom of the test tube to the top end of the closed C-shaped clamp is 5mm, the thickness of the wall of the test tube is 0.5mm in the range from 5-15mm upwards to 5-15mm downwards, the thickness of the wall of other areas is 1-2mm, and the thicknesses of the junctions of different wall thicknesses are gradually changed.
The circumferential section of the thimble is L-shaped, and the L-shaped surface acts on the root of the C-shaped clamp.
The biological fluid sampling and separating method capable of realizing solid-liquid phase isolation after centrifugation comprises the following specific steps,
step one: the construction, collection and separation device comprises a test tube, a guide rail shell, a thimble ring, a clamp ring, a rotary tooth column, a thrust tooth column, a push rod and a spring,
the test tube is of a thick structure at two thin ends in the middle, one end of an opening of the test tube is sleeved on the small diameter of the inner cavity of the test tube connecting ring, the spring is arranged in a cavity formed by the outer wall of the test tube and the test tube connecting ring, one end of the guide rail shell is fixedly connected with the large diameter end of the inner cavity of the test tube connecting ring, and one end of the push rod can enter from the other end of the guide rail shell;
the guide rail shell is of a cylindrical structure with a cavity, clamping grooves are uniformly arranged on the inner wall of the guide rail shell, which is close to one end of the test tube connecting ring, along the axial circumferential direction, and protruding guide rails are uniformly arranged on the inner wall of the guide rail shell, which is close to one end of the push rod, along the axial circumferential direction;
the thimble ring, the clamp ring, the rotary tooth column and the thrust tooth column are sleeved on the test tube in sequence and are arranged in the inner cavity of the guide rail shell;
the thimble ring is of a hollow cavity structure, the thimble which is symmetrically designed is fixedly connected with one end of the thimble ring which is close to one end of the test tube connecting ring along the axial circumferential direction,
the clamp ring is coaxially sleeved on the thimble ring, the clamp ring is fixedly connected with the guide rail shell, and the thimble ring and the clamp ring can slide relatively along the circumferential direction; the clamp ring is axially provided with a thimble groove matched with the thimble, the inner wall of the clamp ring is symmetrically connected with a C-shaped clamp, and the initial state of the C-shaped clamp is in an open ring shape;
the rotary tooth column is of a hollow column structure, the outer wall of the rotary tooth column is uniformly provided with rotary tooth column grooves along the axial circumferential direction, one end of the rotary tooth column is coaxially contacted with the other end of the thimble ring,
the thrust tooth column is of a hollow column structure, the outer wall of the thrust tooth column is uniformly provided with thrust tooth column grooves along the axial circumferential direction, the thrust tooth column grooves are always matched with the convex guide rail of the guide rail shell, one end of the thrust tooth column is in staggered engagement with the other end of the rotary tooth column, and the other end of the thrust tooth column is in contact with one end of the push rod;
step two: the device is placed into a centrifugal machine for centrifugation after a biological fluid sample is collected, a solid phase and a liquid phase are separated under the action of centrifugal force, a push rod is pushed under the action of the centrifugal force during centrifugation, the push rod acts on a thrust tooth column, the thrust tooth column axially moves along a convex guide rail of a guide rail shell, the acting force of a spring is overcome, and the thrust tooth column drives a rotary tooth column and a thimble ring to synchronously move;
step three: when the tooth tips of the rotary tooth columns exceed the raised guide rails, the rotary tooth columns rotate under the action of spring force and guided by the inclined planes at the top ends of the tooth columns until the tooth surfaces of the two tooth columns are meshed, and the C-shaped clamp is in an open state at the moment, so that the effect of centrifugally separating solid and liquid phases is not influenced;
step four: when the centrifugation is finished, the rotation speed is reduced, the centrifugal force is gradually reduced, the spring force is gradually larger than the centrifugal force, the spring force pushes the thimble ring, so that the thimble ring pushes the rotary tooth column to slide into the convex guide rail and move reversely along the convex guide rail, and therefore, under the action of the spring force, the thimble ring, the rotary tooth column and the thrust tooth column move reversely together, the thimble of the thimble ring is placed in the thimble groove of the clamp ring, and meanwhile, the thimble acts on the C-shaped clamp of the clamp ring, so that the closing of the two C-shaped clamps is realized, the fluid channel of the test tube is closed, and the automatic solid-liquid phase physical isolation is realized;
step five: taking out the isolated upper layer liquid phase, if the lower layer solid phase is also taken, the upper layer liquid phase can be centrifuged again, or the push rod is directly pressed by force until the tooth tip of the rotary tooth column exceeds the convex guide rail and then loosened, so that the thimble ring can be withdrawn from the thimble groove, the test tube pipeline is opened, and the lower layer solid phase is taken out.
Compared with the prior art, the invention has the following beneficial effects:
(1) The device of the invention skillfully utilizes the elasticity and centrifugal force of the spring, can ensure the solid-liquid phase physical isolation of various biological fluid samples such as blood and the like through the centrifugation by the test tube size and the design of the C-shaped clamp position, and the liquid phase part after the isolation is completely free of solid matters, thus avoiding the cfDNA existing in the liquid phase from being polluted by the genomic DNA released by cells; the cfDNA protection reagent is matched, so that the cfDNA can be stably stored and transported within the range of-80 ℃ to 40 ℃ for more than 2 weeks;
(2) All cfDNA automatic extraction at present is an automatic extraction process which can only be started after a liquid phase sample existing in cfDNA is manually separated and taken out and then added into an instrument sample cell, so that the efficiency is low when a large number of samples are extracted, and mutual pollution among samples is easily caused by manual misoperation. The device realizes automatic solid-liquid separation after centrifugation, can replace the manual separation sampling process by being provided with the automatic liquid suction needle and other devices, provides great possibility for starting the cfDNA automatic extraction process after sampling and centrifugation, reduces direct contact between an experimenter and a biological sample, and is also a protection for the experimenter.
Drawings
FIG. 1 is a schematic diagram of the complete structure of the present invention;
FIG. 2 is a schematic diagram of the semi-sectional structure of the test tube of the invention in scheme 1 and scheme 2;
FIG. 3 is a schematic diagram of the semi-section structure of a test tube solution 3 according to the present invention;
FIG. 4 is a schematic diagram of the test tube scheme 3 of the present invention;
FIG. 5 is an exploded view of the thimble ring and the clamp ring of the present invention;
FIG. 6 is a schematic view of an assembly of a thimble ring and a clamp ring according to the present invention;
FIG. 7 is a structural outline view of the guide rail housing of the present invention;
FIG. 8 is a half cross-sectional view of a guide rail housing of the present invention;
FIG. 9 is an interior view of the track housing of the present invention;
FIG. 10 is an exploded view of the rotary tooth post, thrust tooth post and pushrod of the present invention;
FIG. 11 is a schematic view of a clamp ring structure of the present invention;
fig. 12 is an overall outline view of the structure of the present invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings.
The automatic isolating device after biological fluid sample collection and centrifugation delamination designed by the invention can conveniently realize solid-liquid phase physical separation (such as separation of blood cells and plasma) of the sample, and the physical isolation can effectively block cfDNA in a cell genome DNA polluted liquid phase layer. The cfDNA component in various body fluid samples under all extreme temperature conditions can be stably stored and transported by matching with the DNA protective agent. Taking a blood sample as an example, the physical separation of blood cells and plasma can be automatically realized after blood centrifugation and delamination, and the traditional plasma separation operation which is performed by manually opening a cover can be released. This has 3 outstanding advantages, 1) direct contact between the experimenter and the biological sample is avoided, and the experimenter is protected, and meanwhile, errors possibly caused by manual operation are avoided; 2) After automatic isolation, cfDNA exists in a liquid phase, so that the cfDNA is completely prevented from being polluted by cell genome DNA; 3) Automatic solid-liquid isolation provides a prerequisite for full automation of cfDNA extraction. The cfDNA protection reagent is matched, so that the cfDNA can be stably stored and transported within the range of-80 ℃ to 40 ℃ for more than 2 weeks.
The specific scheme is as follows: a biological fluid sampling and separating device capable of realizing solid-liquid phase isolation after centrifugation, as shown in figures 1 and 12, comprises a test tube 1, a guide rail shell 2, an ejector ring 3, a clamp ring 4, a rotary tooth column 5, a thrust tooth column 6, a push rod 7 and a spring 8,
the test tube 1 is of a thick structure at two ends with a thin middle part, the thin middle part can realize that the wall thickness of a middle hose part is smaller when the inner diameters are the same, and the test tube is easy to be clamped by the C-shaped clamp 12 to realize the physical blocking of a solid-liquid phase layer; the thick wall thickness at both ends of the test tube is ensured, so that the rigidity of the test tube is increased. One end of the opening of the test tube 1 is sleeved on the small diameter of the inner cavity of the test tube connecting ring 11, the spring 8 is arranged in a cavity formed by the outer wall of the test tube 1 and the test tube connecting ring 11, one end of the guide rail shell 2 is fixedly connected with the large diameter end of the inner cavity of the test tube connecting ring 11, and one end of the push rod 7 can enter from the other end of the guide rail shell 2;
as shown in fig. 7, 8 and 9, the guide rail housing 2 has a cylindrical structure with a cavity, and clamping grooves 9 are uniformly arranged on the inner wall of the guide rail housing 2 near one end of the test tube connecting ring 11 along the axial circumferential direction, and the clamping grooves 9 are preferably 4, have a certain inclination, are engaged with the clamping convex grooves on the periphery of the clamp ring 4 in a complementary manner, and can ensure that the clamp ring 4 is accurately positioned after being assembled into the guide rail housing. The inner wall of the guide rail shell 2 near one end of the push rod 7 is uniformly provided with a convex guide rail 10 along the axial circumferential direction;
the thimble ring 3, the clamp ring 4, the rotary tooth column 5 and the thrust tooth column 6 are sleeved on the test tube 1 in sequence and are arranged in the inner cavity of the guide rail shell 2; the precise matching of the convex guide rail 10 with the grooves on the outer walls of the rotary tooth column 5 and the thrust tooth column 6 is the key for realizing the clamping and the loosening of the C-shaped clamp 12 under the action of centrifugal force and spring force. One end of the thrust tooth column is in staggered engagement with the other end of the rotary tooth column, and the other end of the thrust tooth column is contacted with one end of the push rod and can be pushed by the push rod; the grooves of the thrust tooth columns 6 are not separated from the convex guide rails all the time, and the grooves of the rotary tooth columns 5 can alternately slide into the convex guide rails and separate from the convex guide rails to be clamped by the convex guide rails under the pushing of the thrust tooth columns 6 so as to clamp and unclamp the C-shaped clamp 12.
As shown in fig. 5 and 6, the thimble ring 3 has a hollow cavity structure, and a symmetrically designed thimble is fixedly connected to one end of the thimble ring 3 near one end of the test tube connecting ring 11 along the axial circumferential direction, and the thimble 13 has an L-shaped periphery Xiang Jiemian and an L-shaped surface acting on the root of the C-shaped clamp 12.
The clamp ring 4 is coaxially sleeved on the thimble ring 3, as shown in fig. 11, the clamp ring 4 is fixedly connected with the guide rail shell 2, and the thimble ring 3 and the clamp ring 4 can slide relatively along the circumferential direction; the clamp ring 4 is axially provided with a thimble groove matched with the thimble, the inner wall of the clamp ring 4 is symmetrically connected with a C-shaped clamp 12, the initial state of the C-shaped clamp 12 is in an open ring shape, and the C-shaped clamp 12 is made of metal and does not deform. The design of thimble ring 3, anchor clamps ring 4 and C anchor clamps has effectively practiced thrift the space when guaranteeing to realize pressing from both sides tight test tube and cut off the function of solid-liquid phase layer, makes whole device keep compact structure.
As shown in fig. 10, the rotary tooth column 5 is of a hollow cylindrical structure, the outer wall of the rotary tooth column 5 is uniformly provided with grooves along the axial circumferential direction, one end of the rotary tooth column 5 is coaxially contacted with the other end of the thimble ring 3, and the distance between the thimble in the initial state and the C-shaped clamp 12 is smaller than the distance of the backward movement of the following rotary tooth column 5.
The thrust tooth post 6 is a cavity column structure, the outer wall of the thrust tooth post 6 is uniformly provided with thrust tooth post 6 grooves along the axial circumferential direction, the thrust tooth post 6 grooves are always matched with the convex guide rail 10 of the guide rail shell 2, one end of the thrust tooth post 6 is in dislocation engagement with the other end of the rotary tooth post 5, and the other end of the thrust tooth post 6 is in contact with one end of the push rod 7.
When in operation, the device comprises: when the device after the biological fluid sample is collected is placed into a centrifugal machine to be centrifuged under the centrifugal force of 1200g-2000g, a solid phase (such as blood cells) and a liquid phase (such as blood plasma) are separated under the action of the centrifugal force, meanwhile, under the action of the centrifugal force, a thrust tooth column 6 moves axially along a convex guide rail 10 of a guide rail shell 2, the acting force of a spring 8 is overcome, the thrust tooth column 6 drives a rotary tooth column 5 and a thimble ring 3 to synchronously move, when the tooth tip of the rotary tooth column 5 exceeds the convex guide rail 10, the rotary tooth column 5 rotates under the action of the elastic force of the spring 8 and under the guidance of the inclined plane of the top end of the tooth column, and a C-shaped clamp 12 is in an open state at the moment, so that the centrifugal separation effect is not influenced at all; when the centrifugation is finished, the rotation speed is reduced, the centrifugal force is gradually reduced, the elastic force of the spring 8 is gradually larger than the centrifugal force, the elastic force can push the thimble ring 3, so that the thimble ring 3 pushes the rotary tooth column 5 to slide into the convex guide rail 10 and move reversely along the convex guide rail 10, and therefore under the action of the force of the spring 8, the thimble ring 3, the rotary tooth column 5 and the thrust tooth column 6 move reversely together, the thimble of the thimble ring 3 is arranged in the thimble groove of the clamp ring 4, and meanwhile, the thimble acts on the C-shaped clamp 12 of the clamp ring 4, so that the closing of the two C-shaped clamps 12 is realized, the fluid passage of the test tube 1 is closed, and the automatic solid-liquid phase physical isolation is realized.
The test tube 1 is a combination of plastic materials with different hardness, and comprises any one of the following schemes:
as shown in fig. 2, scheme 1: the range from 5-15mm (preferably 10 mm) upwards of the C-shaped clamp 12 to 5-15mm (preferably 10 mm) downwards of the C-shaped clamp 12 is soft plastic, and the Shore A hardness is 50-70; the other parts of the test tube 1 are made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring 11 and the test tube 1 are an injection molding whole;
scheme 2: the range from the bottom of the test tube 1 to the C-shaped clamp 12 upwards is 5-15mm (preferably 10 mm), the range is soft plastic, and the Shore A hardness is 50-70; the other parts of the test tube 1 are made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring 11 and the test tube 1 are an injection molding whole;
as shown in fig. 3 and 4, scheme 3: the inner core of the test tube 1 is made of soft plastic, the Shore hardness A is 50-70, the test tube connecting ring 11 sleeved at one end of the opening of the test tube 1 is made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring 11 and the test tube 1 are tightly clamped and glued into a whole through the outer wall structure at one end of the opening of the test tube 1.
The whole length of the test tube 1 is 9-13mm, the inner diameter of the test tube 1 is 7-11mm, the distance from the bottom of the test tube 1 to the top end of the closed C-shaped clamp 12 is 3-8mm, and the wall thickness of the test tube 1 is 0.5-2mm.
Preferably, the whole length of the test tube 1 is 11mm, the inner diameter of the test tube 1 is 9mm, the distance from the bottom of the test tube 1 to the top end of the closed C-shaped clamp 12 is 5mm, the range from the upward direction of the C-shaped clamp 12 to the downward direction of the C-shaped clamp 12 is 5-15mm (preferably 10 mm), the wall thickness of the test tube 1 is 0.5mm, the wall thickness of other areas is 1-1.5mm, and the junction of different wall thicknesses is subjected to thickness gradual change treatment.
The biological fluid sampling and separating method for realizing solid-liquid phase isolation after centrifugation by using the device comprises the following specific steps:
step one: constructing the acquisition and separation device;
step two: the device is put into a centrifugal machine for centrifugation after the biological fluid sample is collected, solid phase and liquid phase are separated under the action of centrifugal force, when the device is centrifuged, a push rod 7 is pushed under the action of the centrifugal force, the push rod 7 acts on a thrust tooth column 6, the thrust tooth column 6 moves axially along a convex guide rail 10 of a guide rail shell 2, the acting force of a spring 8 is overcome, and the thrust tooth column 6 drives a rotary tooth column 5 and a thimble ring 3 to synchronously move;
step three: when the tooth tips of the rotary tooth columns 5 exceed the raised guide rail 10, the rotary tooth columns 5 rotate under the action of the elasticity of the springs 8 and the guidance of the inclined planes at the top ends of the tooth columns until the tooth surfaces of the two tooth columns are meshed, and at the moment, the C-shaped clamp 12 is in an open state, so that the effect of centrifugal separation of solid and liquid phases is not influenced;
step four: when the centrifugation is finished, the rotation speed is reduced, the centrifugal force is gradually reduced, the elastic force of the spring 8 is gradually larger than the centrifugal force, the spring 8 pushes the thimble ring 3, so that the thimble ring 3 pushes the rotary tooth column 5 to slide into the convex guide rail 10 and move reversely along the convex guide rail 10, and therefore, under the action of the force of the spring 8, the thimble ring 3, the rotary tooth column 5 and the thrust tooth column 6 move reversely together, the thimble of the thimble ring 3 is placed in the thimble groove of the clamp ring 4, and meanwhile, the thimble acts on the C-shaped clamp 12 of the clamp ring 4, so that the closing of the two C-shaped clamps 12 is realized, the fluid passage of the test tube 1 is closed, and the automatic solid-liquid phase physical isolation is realized;
step five: taking out the isolated upper layer liquid phase to carry out relevant detection experiments such as free DNA extraction and the like, taking out the lower layer solid phase, centrifuging again, or directly pressing the push rod 7 with force until the tooth tip of the rotary tooth column 5 exceeds the raised guide rail 10 and then loosening, so that the thimble ring 3 can be withdrawn out of the thimble groove, the tube of the test tube 1 is opened, and taking out the lower layer solid phase.
By using the device disclosed by the invention, the physical isolation between the solid phase and the liquid phase layer can be automatically realized after the biological fluid sample containing solid phase particles is subjected to centrifugal solid-liquid phase layering, so that target detection substances such as free DNA in the liquid phase layer are protected from being polluted by solid phase substances such as cells in the sample preservation and transportation processes. The device can also pump the inner test tube 1 into negative pressure, so as to realize the negative pressure quantitative collection of the sample. The device can be applied to (but not limited to) collection and solid-liquid separation of body fluid samples such as blood, urine, hydrothorax and ascites, milk and the like.
The device of the invention skillfully utilizes the elasticity and centrifugal force of the spring 8, and can ensure that after solid-liquid phase layered isolation is realized through centrifugation through the size of the test tube 1 and the position design of the C-shaped clamp 12, solid-phase substances do not exist in the liquid phase completely, thus avoiding the cfDNA existing in the liquid phase from being polluted by cell genome DNA; the cfDNA protection reagent is matched, so that the cfDNA can be stably stored and transported within the range of-80 ℃ to 40 ℃ for more than 2 weeks;
all cfDNA automatic extraction at present is an automatic extraction process which can only be started after a liquid phase sample existing in cfDNA is manually separated and taken out and then added into an instrument sample cell, so that the efficiency is low when a large number of samples are extracted, and mutual pollution among samples is easily caused by manual misoperation. The device realizes automatic solid-liquid separation after centrifugation, can replace the manual separation sampling process by being provided with the automatic liquid suction needle and other devices, provides great possibility for starting the cfDNA automatic extraction process after sampling and centrifugation, reduces direct contact between an experimenter and a biological sample, and is also a protection for the experimenter.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.
What is not described in detail in the present specification belongs to the known technology of those skilled in the art.
Claims (8)
1. The biological fluid sampling and separating device capable of realizing solid-liquid phase isolation after centrifugation is characterized by comprising a test tube (1), a guide rail shell (2), an ejector ring (3), a clamp ring (4), a rotary tooth column (5), a thrust tooth column (6), a push rod (7) and a spring (8),
the test tube (1) is of a middle thin two-end thick structure, one end of an opening of the test tube (1) is sleeved on the small diameter of the inner cavity of the test tube connecting ring (11), the spring (8) is arranged in a cavity formed by the outer wall of the test tube (1) and the test tube connecting ring (11), one end of the guide rail shell (2) is fixedly connected with the large diameter end of the inner cavity of the test tube connecting ring (11), and one end of the push rod (7) can enter from the other end of the guide rail shell (2);
the guide rail shell (2) is of a cylindrical structure with a cavity, clamping grooves (9) are uniformly distributed on the inner wall of the guide rail shell (2) close to one end of the test tube connecting ring (11) along the axial circumferential direction, and the clamping grooves (9) of the guide rail shell (2) are in complementary engagement with the clamping convex grooves on the periphery of the clamp ring (4);
protruding guide rails (10) are uniformly arranged on the inner wall of the guide rail shell (2) close to one end of the push rod (7) along the axial circumferential direction;
the thimble ring (3), the clamp ring (4), the rotary tooth column (5) and the thrust tooth column (6) are sleeved on the test tube (1) in sequence and are arranged in the inner cavity of the guide rail shell (2);
the thimble ring (3) is a hollow cavity structure, thimble which is symmetrically designed is fixedly connected with one end of the thimble ring (3) which is close to one end of the test tube connecting ring (11) along the axial circumferential direction,
the clamp ring (4) is coaxially sleeved on the thimble ring (3), the clamp ring (4) is fixedly connected with the guide rail shell (2), and the thimble ring (3) and the clamp ring (4) can slide relatively along the circumferential direction; the clamp ring (4) is axially provided with a thimble groove matched with a thimble, the inner wall of the clamp ring (4) is symmetrically connected with C-shaped clamps (12), and the initial state of the C-shaped clamps (12) is in an open ring shape;
the rotary tooth column (5) is of a hollow cylindrical structure, grooves of the rotary tooth column (5) are uniformly formed on the outer wall of the rotary tooth column along the axial circumferential direction, the grooves of the rotary tooth column (5) can alternately slide into and separate from the convex guide rail, one end of the rotary tooth column (5) is coaxially contacted with the other end of the thimble ring (3),
the thrust tooth column (6) is of a hollow column structure, the outer wall of the thrust tooth column is uniformly provided with thrust tooth column (6) grooves along the axial circumferential direction, the thrust tooth column (6) grooves are always matched with the convex guide rail of the guide rail shell (2), one end of the thrust tooth column (6) is in staggered engagement with the other end of the rotary tooth column (5), and the other end of the thrust tooth column (6) is in contact with one end of the push rod (7);
when in operation, the device comprises: the device is placed into a centrifugal machine for centrifugation after a biological fluid sample is collected, a solid phase and a liquid phase are separated under the action of centrifugal force, meanwhile, under the action of the centrifugal force, a thrust tooth column (6) moves axially along a convex guide rail of a guide rail shell (2), the acting force of a spring (8) is overcome, the thrust tooth column (6) drives a rotary tooth column (5) and a thimble ring (3) to move synchronously, when the tooth tip of the rotary tooth column (5) exceeds a convex guide rail (10), the rotary tooth column (5) rotates under the action of the elastic force of the spring (8) and the guide of the inclined plane of the top end of the tooth column until the tooth surfaces of the two tooth columns are meshed, and at the moment, a C-shaped clamp (12) is in an open state; when the centrifugation is finished, the rotation speed is reduced, the centrifugal force is gradually reduced, the elastic force of the spring (8) is gradually larger than the centrifugal force, the elastic force pushes the thimble ring (3), the thimble ring (3) pushes the rotary tooth column (5) to slide into the convex guide rail (10) and move reversely along the convex guide rail (10), the thimble ring (3), the rotary tooth column (5) and the thrust tooth column (6) move reversely together, the thimble of the thimble ring (3) is arranged in the thimble groove of the clamp ring (4), and meanwhile, the thimble acts on the C-shaped clamp (12) of the clamp ring (4) to close the two C-shaped clamps (12), so that the liquid flow channel of the test tube (1) is closed, and automatic solid-liquid phase physical isolation is realized;
the circumferential section of the thimble (13) is L-shaped, and the L-shaped surface acts on the root of the C-shaped clamp (12).
2. The biological fluid sampling and separating device capable of realizing solid-liquid phase separation after centrifugation according to claim 1, wherein the distance from the thimble in the initial state to the C-shaped clamp (12) is smaller than the distance from the subsequent rotary tooth column (5) to move reversely.
3. The biological fluid sampling and separating device capable of realizing solid-liquid phase separation after centrifugation according to claim 1, wherein the test tube (1) is a combination of plastic materials with different hardness, and the device comprises any one of the following schemes:
scheme 1: the range from 5-15mm upwards of the C-shaped clamp (12) to 5-15mm downwards of the C-shaped clamp (12) is soft plastic, and the Shore hardness A is 50-70; the other parts of the test tube (1) are made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring (11) and the test tube (1) are an injection molding whole;
scheme 2: the range from the bottom of the test tube (1) to the upward 5-15mm of the C-shaped clamp (12) is soft plastic, and the Shore A hardness is 50-70; the other parts of the test tube (1) are made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring (11) and the test tube (1) are an injection molding whole;
scheme 3: the inner core of the test tube (1) is made of soft plastic, the Shore hardness A is 50-70, the test tube connecting ring (11) sleeved at one end of the opening of the test tube (1) is made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring (11) and the test tube (1) are tightly clamped and glued into a whole through the outer wall structure at one end of the opening of the test tube (1).
4. The biological fluid sampling and separating device capable of realizing solid-liquid phase separation after centrifugation according to claim 1, wherein the whole length of the test tube (1) is 9-13mm, the inner diameter of the test tube (1) is 7-11mm, the distance from the bottom of the test tube (1) to the top end of the closed C-shaped clamp (12) is 3-8mm, and the wall thickness of the test tube (1) is 0.5-2.5mm.
5. The biological fluid sampling and separating device capable of realizing solid-liquid phase separation after centrifugation according to claim 1, wherein the whole length of the test tube (1) is 11mm, the inner diameter of the test tube (1) is 9mm, the distance from the bottom of the test tube (1) to the top end of the closed C-shaped clamp (12) is 5mm, the wall thickness of the test tube (1) is 0.5mm in the range from 5-15mm upwards to 5-15mm downwards of the C-shaped clamp (12), the wall thickness of other areas is 1-2mm, and the thickness of the junction of different wall thicknesses is gradually changed.
6. The biological fluid sampling and separating method capable of realizing solid-liquid phase separation after centrifugation is characterized by comprising the following specific steps of,
step one: the construction, collection and separation device comprises a test tube (1), a guide rail shell (2), an ejector ring (3), a clamp ring (4), a rotary tooth column (5), a thrust tooth column (6), a push rod (7) and a spring (8),
the test tube (1) is of a middle thin two-end thick structure, one end of an opening of the test tube (1) is sleeved on the small diameter of the inner cavity of the test tube connecting ring (11), the spring (8) is arranged in a cavity formed by the outer wall of the test tube (1) and the test tube connecting ring (11), one end of the guide rail shell (2) is fixedly connected with the large diameter end of the inner cavity of the test tube connecting ring (11), and one end of the push rod (7) can enter from the other end of the guide rail shell (2);
the guide rail shell (2) is of a cylindrical structure with a cavity, clamping grooves (9) are uniformly distributed on the inner wall of the guide rail shell (2) close to one end of the test tube connecting ring (11) along the axial circumferential direction, and raised guide rails (10) are uniformly distributed on the inner wall of the guide rail shell (2) close to one end of the push rod (7) along the axial circumferential direction;
the thimble ring (3), the clamp ring (4), the rotary tooth column (5) and the thrust tooth column (6) are sleeved on the test tube (1) in sequence and are arranged in the inner cavity of the guide rail shell (2);
the thimble ring (3) is a hollow cavity structure, thimble which is symmetrically designed is fixedly connected with one end of the thimble ring (3) which is close to one end of the test tube connecting ring (11) along the axial circumferential direction,
the clamp ring (4) is coaxially sleeved on the thimble ring (3), the clamp ring (4) is fixedly connected with the guide rail shell (2), and the thimble ring (3) and the clamp ring (4) can slide relatively along the circumferential direction; the clamp ring (4) is axially provided with a thimble groove matched with a thimble, the inner wall of the clamp ring (4) is symmetrically connected with C-shaped clamps (12), and the initial state of the C-shaped clamps (12) is in an open ring shape;
the rotary tooth column (5) is of a hollow column structure, grooves of the rotary tooth column (5) are uniformly formed on the outer wall of the rotary tooth column along the axial circumferential direction, one end of the rotary tooth column (5) is coaxially contacted with the other end of the thimble ring (3),
the thrust tooth column (6) is of a hollow cylindrical structure, the outer wall of the thrust tooth column is uniformly provided with thrust tooth column (6) grooves along the axial circumferential direction, the thrust tooth column (6) grooves are always matched with the convex guide rail (10) of the guide rail shell (2), one end of the thrust tooth column (6) is in staggered engagement with the other end of the rotary tooth column (5), and the other end of the thrust tooth column (6) is in contact with one end of the push rod (7);
step two: the device is placed into a centrifugal machine for centrifugation after a biological fluid sample is collected, a solid phase and a liquid phase are separated under the action of centrifugal force, a push rod (7) is pushed under the action of the centrifugal force during centrifugation, the push rod (7) acts on a thrust tooth column (6), the thrust tooth column (6) moves axially along a convex guide rail (10) of a guide rail shell (2), the acting force of a spring (8) is overcome, and the thrust tooth column (6) drives a rotary tooth column (5) and a thimble ring (3) to move synchronously;
step three: when the tooth tip of the rotary tooth column (5) exceeds the raised guide rail (10), the rotary tooth column (5) rotates under the elastic action of the spring (8) and the guide of the inclined plane at the top end of the tooth column until the tooth surfaces of the two tooth columns are meshed, and at the moment, the C-shaped clamp (12) is in an open state, so that the effect of centrifugal separation of solid and liquid phases is not influenced;
step four: when the centrifugation is finished, the rotation speed is reduced, the centrifugal force is gradually reduced, the elastic force of the spring (8) is gradually larger than the centrifugal force, the elastic force of the spring (8) pushes the thimble ring (3), so that the thimble ring (3) pushes the rotary tooth column (5) to slide into the convex guide rail (10) and move reversely along the convex guide rail (10), and thus, under the action of the force of the spring (8), the thimble ring (3), the rotary tooth column (5) and the thrust tooth column (6) move reversely together, the thimble of the thimble ring (3) is placed in the thimble groove of the clamp ring (4), and meanwhile, the thimble acts on the C-shaped clamp (12) of the clamp ring (4), so that the closing of the two C-shaped clamps (12) is realized, the flow channel of the test tube (1) is closed, and the automatic solid-liquid phase physical isolation is realized;
step five: taking out the isolated upper liquid phase, if the lower solid phase is also taken, the upper liquid phase can be centrifuged again, or the push rod (7) can be directly pressed by force until the tooth tip of the rotary tooth column (5) exceeds the convex guide rail (10) and then loosened, so that the thimble ring (3) can be withdrawn from the thimble groove, the tube of the test tube (1) is opened, and the lower solid phase is taken out.
7. The biological fluid sampling and separating method capable of realizing solid-liquid phase separation after centrifugation according to claim 6, wherein the test tube (1) is a combination of plastic materials with different hardness, and the method comprises any one of the following schemes:
scheme 1: the range from 5-15mm upwards of the C-shaped clamp (12) to 5-15mm downwards of the C-shaped clamp (12) is soft plastic, and the Shore hardness A is 50-70; the other parts of the test tube (1) are made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring (11) and the test tube (1) are an injection molding whole;
scheme 2: the range from the bottom of the test tube (1) to the upward 5-15mm of the C-shaped clamp (12) is soft plastic, and the Shore A hardness is 50-70; the other parts of the test tube (1) are made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring (11) and the test tube (1) are an injection molding whole;
scheme 3: the inner core of the test tube (1) is made of soft plastic, the Shore hardness A is 50-70, the test tube connecting ring (11) sleeved at one end of the opening of the test tube (1) is made of hard plastic, the Shore hardness D is 70-80, and the test tube connecting ring (11) and the test tube (1) are tightly clamped and glued into a whole through the outer wall structure at one end of the opening of the test tube (1).
8. The method for sampling and separating biological fluid capable of realizing solid-liquid phase separation after centrifugation according to claim 6, wherein the whole length of the test tube (1) is 9-13mm, the inner diameter of the test tube (1) is 7-11mm, and the distance from the bottom of the test tube (1) to the top end of the closed C-shaped clamp (12) is 3-8mm.
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