CN113834799A - Separation system for target substance in biological liquid sample - Google Patents
Separation system for target substance in biological liquid sample Download PDFInfo
- Publication number
- CN113834799A CN113834799A CN202110861683.XA CN202110861683A CN113834799A CN 113834799 A CN113834799 A CN 113834799A CN 202110861683 A CN202110861683 A CN 202110861683A CN 113834799 A CN113834799 A CN 113834799A
- Authority
- CN
- China
- Prior art keywords
- membrane
- vibration
- cells
- separation system
- support plate
- 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.)
- Pending
Links
- 238000000926 separation method Methods 0.000 title claims abstract description 89
- 239000013076 target substance Substances 0.000 title claims abstract description 34
- 239000007788 liquid Substances 0.000 title claims abstract description 31
- 239000012528 membrane Substances 0.000 claims abstract description 191
- 230000005540 biological transmission Effects 0.000 claims abstract description 10
- 239000013060 biological fluid Substances 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 99
- 239000011148 porous material Substances 0.000 claims description 23
- 210000004881 tumor cell Anatomy 0.000 claims description 9
- 239000000427 antigen Substances 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 241000700605 Viruses Species 0.000 claims description 2
- 230000002159 abnormal effect Effects 0.000 claims description 2
- 210000001789 adipocyte Anatomy 0.000 claims description 2
- 230000002538 fungal effect Effects 0.000 claims description 2
- 210000004698 lymphocyte Anatomy 0.000 claims description 2
- 210000002540 macrophage Anatomy 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 claims description 2
- 230000000735 allogeneic effect Effects 0.000 claims 1
- 210000003040 circulating cell Anatomy 0.000 claims 1
- 230000006378 damage Effects 0.000 abstract description 9
- 230000004071 biological effect Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 33
- 230000000694 effects Effects 0.000 description 21
- 239000000126 substance Substances 0.000 description 15
- 229920000052 poly(p-xylylene) Polymers 0.000 description 14
- 238000001914 filtration Methods 0.000 description 11
- 239000012472 biological sample Substances 0.000 description 9
- 239000004642 Polyimide Substances 0.000 description 8
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 8
- 230000028161 membrane depolarization Effects 0.000 description 8
- 239000012982 microporous membrane Substances 0.000 description 8
- 229920001721 polyimide Polymers 0.000 description 8
- 230000004907 flux Effects 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000009471 action Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000004417 polycarbonate Substances 0.000 description 4
- 229920000515 polycarbonate Polymers 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 230000005484 gravity Effects 0.000 description 3
- 230000033001 locomotion Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000004492 nuclear pore Anatomy 0.000 description 2
- 230000010287 polarization Effects 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000006735 deficit Effects 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
- 238000005530 etching Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000009285 membrane fouling Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 229910052755 nonmetal Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000000206 photolithography Methods 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- -1 polyethylene terephthalate Polymers 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Pathology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Sustainable Development (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention provides a separation system for a target substance in a biological fluid sample, the system comprising: a microwell array membrane with which a biological fluid sample is received or contacted; a vibration power source component capable of generating vibration, and a transmission component for transmitting the vibration generated by the vibration power source to the micropore array membrane. By using the system, the target substance in the biological liquid can be separated out efficiently through the membrane, and the biological activity of the target substance is kept to the maximum extent without damage.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a system and a method for separating special cells in a sample by using a microporous array filter membrane.
Background
The microporous array filter membrane with high porosity is suitable for being applied to concentration and separation of clinical or biological liquid samples. Although microporous array filtration membranes generally have higher porosity and lower transmembrane resistance than traditional chemical membranes, in general, clogging is less likely to occur during moderate amounts of clinical and biological fluid sample separation.
However, when the number of cells or particles in the liquid sample far exceeds the number of micropores, a congestion situation inevitably occurs, and even the filtration cannot be completed due to complete pore blocking. In practical application, the number of white blood cells in 1mL of normal human blood is 400-1 million, the number of red blood cells is more than one hundred million, and if the number of micropores in a working area is far lower than the number of cells, especially when the number of target cells intercepted on a membrane exceeds a certain number, certain congestion can occur. After congestion occurs, not only the time for cell separation is multiplied, but also the separation accuracy and screening size are affected by the change of the micro-flow field due to the accumulation of "membrane fouling", which is understood as the accumulation of other non-target substances, such as white blood cells or red blood cells. Therefore, in practical application, a scheme capable of effectively improving separation flux and relieving congestion is needed.
Disclosure of Invention
In order to improve the separation effect of a high-flux microporous array membrane on a target substance in a biological sample, the invention provides a separation method and a separation system, which can effectively solve some defects of the traditional technology.
In one aspect the invention provides a separation system comprising: a microwell array membrane with which a biological sample is received or contacted; a vibration power source element capable of generating vibration; and a transmission element for transmitting the vibration from the vibration power source to the micropore array membrane.
In some embodiments, the microporous array membrane comprises 500-50000000 micropores per square millimeter in unit area, the diameter of the micropores is 0.1-50 microns, the thickness of the membrane is 1-100 microns, and the porosity is 5% -90%. The micro-processing membrane mainly refers to precise holes from the processing category, and also comprises a part of chemical membrane with narrow size distribution and low membrane resistance and a nuclear pore membrane. The membrane material may be Polycarbonate (PC), Polyimide (PI), Parylene (Parylene), etc., and the Parylene microporous array membrane is preferred in the present invention. The micropore structure can be round, square, rectangle, regular hexagon and the like, and the diameter of the micropore refers to the equivalent diameter corresponding to the inscribed circle of the micropore if no other description exists.
In some forms, the vibration driving power source includes various mechanical vibration generators such as a hollow cup vibration motor and/or an eccentric wheel direct current motor; an ultrasonic generator based on a transducer chip and/or a pneumatic vibration device.
In some forms, the transmission element includes an element capable of stably transmitting vibration to the membrane, such as a spring transmission and a metal rod transmission. The transmission element comprises a support plate, and the spring is arranged below the support plate and used for supporting the support plate, so that the support plate is supported by the spring to stand. In some embodiments, the springs are located below the support plate, with three or more spring elements, allowing the support plate to be in a stable state, but suspended by the springs. One end of the spring element is used for supporting the supporting plate, and the other end of the spring element can be positioned on the operating table top, so that the whole vibration system is in a relatively independent position.
In some embodiments, the membrane is disposed in a membrane carrier container, such that the carrier container includes the membrane and a chamber for receiving the sample, such that the liquid sample in the chamber is in direct contact with the membrane. In some forms, the container includes an ultra-low negative pressure element positioned below the membrane for applying a pressure to the membrane. The pressure applying mode can be various, and can be a vacuum pump or a cavity (lower cavity), wherein liquid is contained in the cavity, and certain pressure is applied by the gravity of the liquid. Of course, it is also possible to accelerate the passage of liquid from the membrane (the liquid sample in the chamber receiving the sample) through the membrane by means of the liquid sample flowing through the membrane into the chamber, thereby creating a negative pressure below the membrane, and thus allowing the sample to pass through the membrane more quickly, and allowing substances that cannot pass through the membrane, such as tumor cells, to rest on the membrane. At this time, the membrane is located between the sample-receiving chamber and the lower chamber.
In some modes, the vibration driving power source is arranged on the support plate or fixedly connected with the support plate, and when the vibration power source vibrates, the vibration can be transmitted to the support plate and then transmitted to the membrane through the support plate. Thereby, the target substance in the biological sample on the membrane is trapped on the membrane, and the non-target substance in the sample flows out through the micropores of the membrane. In some embodiments, the vibration source includes one or two vibration sources, the two vibration sources having different powers.
In some forms, the support plate includes structure thereon for mounting the film carrier container, whereby the film carrier container is removably secured to the support plate. Thus, the membrane carrier container can be removably replaced when the system is in use, so that a variety of different biological samples can be filtered using different membrane carrier containers using the same system. For example, each membrane carrier container filters one biological sample, while multiple membrane carrier containers can filter different biological samples multiple times. In some forms, the film carrier container includes a bottom annular groove, and the support plate has a raised structure that engages the annular groove to secure the film carrier container. In some modes, a limiting block is arranged on the supporting plate and clamped on the container, so that the position of the container is not changed. Thus, in fact, the container and the supporting plate are connected into a whole, and the vibration waves emitted by the vibration source can be conveniently transmitted to the film of the container.
In some embodiments, the target substance in the biological sample is tumor cells, circulating abnormal cells, exfoliated tumor cells, cell lines capable of producing antibodies or antigens, lymphocytes or stem cells of autologous or heterologous origin, adipocytes, macrophages, fungal spores, bacteria, viruses, extracellular vesicles, or other biologically active substances. These substances generally cannot pass through the micropores or sub-micropores of the membrane, while other non-target substances in the sample can pass through the micropores, such as water molecules, cells/particles smaller than the micropores, and cells/particles with stronger deformability, so as to achieve the purpose of filtration and separation.
In some embodiments, the system further comprises an ultra-low pressure separation system for pushing the liquid sample with various cell types and other substances randomly distributed to the surface of the microporous array membrane for the screening based on the membrane surface structure and the pore size under stable ultra-low pressure.
Under the action of a single ultra-low pressure micropore array membrane separation strategy, along with the advance of separation time, non-target substances and target substances are gradually enriched on two sides of the membrane respectively, and the separation or concentration effect is achieved. However, as the filtration proceeds, according to the designed separation strategy, substances on both sides of the membrane accumulate, the order of the total sample is improved, and when a large number of ordered cells or substances accumulate on one side of the membrane and the originally randomly distributed liquid sample cannot effectively contact the membrane, it is generally considered that the sample on the surface of the membrane has a serious "polarization" phenomenon, and thus the order of the sample cannot be further efficiently separated and improved, thereby affecting the separation effect and efficiency of the target substance (fig. 9, the leftmost schematic diagram). In this case, an efficient method is needed for depolarization, i.e., local sample re-randomization. The ultra-low pressure microporous array filter membrane is characterized in that the surface order is far superior to that of the traditional chemical membrane, so that the polarization degree of the local part of the membrane surface to a sample is very high. However, the vibration of the invention just overcomes the inherent defect or shortcoming of the single adoption of the ultra-low pressure micropore array membrane separation strategy, and the high-efficiency local uniform mixing caused by the vibration is a very effective depolarization scheme. Therefore, if the vibration separation method of the present invention is added in the case of ultra low pressure, separation and/or concentration can be performed more rapidly and better.
In some forms the system further comprises a vibration control system for controlling a frequency at which the vibratory drive power source emits vibrations.
In the present invention, after the vibration wave of the rated frequency is emitted by the vibration driving power source, the vibration wave is transmitted to the membrane through the transmission element (the support plate or the support plate with the spring), so that the biological sample on the membrane is vibrated, the vibration vibrates different substances, such as different cells, in the sample, the vibration separates target substances, such as tumor cells, from the surface of the membrane, so that non-target substances, such as water molecules and other cells, pass through the micropores, and only the target substances are on the surface of the membrane, thereby achieving the purpose of separation. In another aspect, the vibration not only drives the target substance to separate from the surface of the membrane, but actually drives the substance on the membrane to move transversely, the transverse position movement is generally difficult to see by naked eyes, the micro displacement is caused by the transmission element, and when the vibration occurs, the support plate and the spring are naturally subjected to micro left-right movement, so that the substance on the membrane is driven to move while separating from the surface of the membrane, and thus, the flow and depolarization of the non-target substance and the target substance can be accelerated, the randomness of the sample on the surface of the membrane is enhanced, the high efficiency of separating the sample on the surface of the membrane is maintained, and the purpose of quickly separating the target substance can be achieved. This is a possible technical principle to explain the present invention in principle. This ensures both the efficiency of the separation and the quality of the separated target substance, e.g.cells, without damage (the rightmost diagram of FIG. 9).
Such a separation principle is different from the principle of depolarization optimization separation using a conventional vortex method. When separation is performed by adopting a vortex mode (the middle schematic diagram of fig. 9), in fact, by rotating centrifugation or eccentric motion, the sample is enabled to produce the effect of vortex mixing uniformly as a whole, if the shear field generated by the vortex relative to the membrane surface is large enough, cells can be separated from the membrane surface, and thus the effect of sample depolarization can also be achieved, but the separation mode usually does not consider the problem of activity of the target substance too much, but removes impurities in the sample. This is because when the liquid is swirled, it is necessary to generate a shear field that can affect the liquid film and cells on the membrane surface, especially cells in the central portion of the membrane, so that the shear field distribution on the membrane is enhanced in the direction from the center to the inner wall end of the cavity (including the entire inner wall of the cavity), which often satisfies the requirement of depolarization of the sample in the central portion of the membrane, and causes excessive shear force damage to the cells in the peripheral region of the membrane and in the vicinity of the inner wall of the cavity, thereby damaging the biological activity of the sample, especially damaging or damaging the cells of the target cells staying on the membrane surface under high shear force, and significantly affecting the activity and the re-culture survival rate of the target cells. Compared with the prior art, the vibration scheme of the invention can form uniform and stable sample depolarization effect on the surface of the membrane, and can more efficiently meet the requirements of blocking prevention and separation efficiency improvement without influencing separation precision and cell activity.
In addition, when the separation of the target substance is carried out in the present invention, the activity of an undesired substance, for example, a tumor cell or a cell line producing an antigen antibody, is affected, for example, a cell stress signal pathway is activated, apoptotic, or even disrupted, and thus, although the separation is achieved, if the separated cell is disrupted or the biological function is affected, the subsequent use cannot be carried out. For example, isolated tumor cells are collected and subsequently cultured and drug-sensitive for other studies. After the antigen-antibody producing cells are isolated, culture is continued to produce antibodies (cell perfusion). Therefore, the frequency of the vibration is not too high or too low, and if it is too low, the separation effect is not achieved or the separation efficiency is low, the time is long, and too high may cause damage to the target cells.
A microporous array membrane: the micropore array membrane of the invention is a micropore array membrane or a chip with precise pores or narrow pore diameter distribution range (within 5%), and the micropores can be one of round holes, squares, hexagons, long strips or irregular shapes. The micropore density is high (the general porosity is more than 20 percent), and the ratio of the thickness of the membrane or the chip to the pore size is not more than 10 times. Such a microporous array membrane includes a chemical membrane (e.g., an alumina separation membrane, a polystyrene separation membrane, etc.) having very precise distribution of micropores, a core pore membrane (e.g., a polyethylene terephthalate microporous membrane, or a polycarbonate microporous membrane, etc.) having relatively uniform distribution of micropores, and more micro-machined membranes prepared by a micro electro mechanical system (MEMS process such as photolithography, etching, imprinting, etc.), including a Polyimide (PI) microporous array membrane, a polyacrylate microporous array membrane, various photosensitive resin microporous array membranes, a parylene microporous array membrane, etc. These membranes can be used by the inventive vibrating device for the analysis of target substances in liquid samples. In some embodiments, a nuclear pore membrane of polycarbonate, a polyimide microwell array membrane, and a parylene microwell array membrane, respectively, were used as test objects. When the mechanical strength of the membrane material can bear the vibration strength, and the pore diameter of the micropores is not particularly close to the size of a target separation object so as to ensure that the interception influence of the material on the target separation object is not large, the effect trends of the various membranes on cell separation under the assistance of the vibration system are consistent, and the separation performance curve can be changed due to the difference between the porosity and the membrane passing resistance, but the advantages of the invention can be embodied on the whole. The parylene microporous array membrane with the optimal performance is stable and efficient in separation under the assistance of a vibration system and small in cell loss. The hexagonal polyimide microporous array membrane and the parylene microporous array membrane have the advantages of optimal application effect on the recovery and re-culture, high separation efficiency and recovery rate and small damage to cells under the same vibration condition and the optimal separation membrane aperture size.
The microporous membrane with the characteristics has uniform and low transmembrane resistance, and is usually operated in an ultralow pressure state (separation pressure does not exceed 100 KPa). The membrane and the liquid sample can be stably relatively displaced by vibration with a certain frequency, so that tangential shear flow is formed, the anti-blocking effect is increased, and the separation precision is not influenced. The shear flow occurs in a very microscopic state around the pores of each membrane, so that the separation and concentration of cells can be realized, and the micro vibration amplitude is also provided, so that the cells can move in a small range, the separation of target cells and non-target substances can be realized, and the separation of bioactive substances can be quickly and effectively carried out in a high-flux manner, particularly the separation of some target cells from clinical or biological samples.
In another aspect, the present invention provides a method of separating a target substance from a biological fluid sample, the method comprising providing a separation system as described above, and causing the source of vibratory power to emit vibratory waves of a frequency such that the target substance is retained on the membrane and non-target substances are allowed to flow through the membrane.
Drawings
Fig. 1 is a schematic perspective view (upright state) of a container provided with a film according to an embodiment of the present invention.
Fig. 2 is a schematic perspective view (upside down state) of a container provided with a film according to an embodiment of the present invention.
Fig. 3 is a cross-sectional view of a container provided with a membrane in accordance with an embodiment of the present invention.
Fig. 4 is a perspective view (front view in assembled state) of a separation system in an embodiment of the invention.
FIG. 5 is a perspective view of a separation system in accordance with one embodiment of the present invention (assembled back)
FIG. 6 is a schematic cross-sectional view of a separation system in accordance with one embodiment of the present invention.
Fig. 7 is a schematic perspective exploded view of a separation system in accordance with an embodiment of the present invention.
FIG. 8 is a graph of the results of the separation of different sizes of microwells at a frequency of 50Hz and the recovery of the residual cells on the final membrane compared to the residual cells on the final membrane without the application of the shaking Filter (Filter-4 indicates a microwell size of 4 microns, followed by 6 microns, 8 microns, 10 microns, 12 microns.).
Fig. 9 is a schematic structural diagram of different separation modes.
Detailed Description
The description of how the present invention may be carried out is by way of example only and is not intended to limit the scope of the invention in any way, without departing from the spirit of the principles of the present invention.
Vibration separation system
The separation system of the present invention, as shown in fig. 4-7, comprises a support plate 60, on which a structure for fixing a membrane carrier container is provided (which may be metal or non-metal), wherein the membrane is a micro-pore array membrane. In some forms, one or more resilient members, preferably four springs 32, 33, 34, 35, are provided beneath the support plate for standing the support plate in a relatively suspended position. One end of the spring may link the support plate and the other end is used to contact the console 40. In some forms, the support plate is provided with a vibratory power source 20 that is capable of inducing vibration when energized. The power source can be fixedly arranged on the supporting plate; for example, as shown in fig. 4, the power source is suspended from the support plate by a suspension structure 201. Of course, a single power source or multiple power sources are possible, with the illustration 606 providing a mounting for additional power sources (small vibration sources). When the vibration motor is one, in order to keep the support plate stable, some counterweight blocks 605 may be added to make the support plate in a relatively stable state in suspension. Of course, other ways may be used to keep the support plate in a relatively stable, suspended state. Thus, when the power source vibrates, energy can be stably transmitted to the membrane through the supporting plate, the spring and the like, and the vibration frequency can be controlled more flexibly and precisely. On the contrary, if the supporting plate contacts many external objects, the vibration wave is weakened or lost in transmission, and finally the vibration wave actually received by the membrane is weak and cannot play a role of depolarization of the membrane surface. In some embodiments, the membrane carrier container may be provided with a hole 603 in the support plate, and the membrane carrier container may be held in the hole by a clamp 30, so that the membrane carrier container is held in the support plate, and the vibration wave from the support plate is stably received, and the liquid in the container passes through the membrane and flows into the lower part of the support plate, and the discharged liquid may be released into the waste liquid collecting container. The clamp 30 can be arranged in various ways, for example, two limit blocks 301,302 matched with the container are arranged at two sides of the container to achieve the function of limiting and fixing. When the container needs to be unloaded, the clamp can be flexibly operated, and the container is convenient to install and unload.
In some embodiments, the film-carrying container 10 generally comprises a chamber 103, the membrane 105 being disposed at the bottom of the chamber, the liquid sample being collected in the chamber 101 above the membrane 105, and the liquid being allowed to flow through the membrane to the other side of the membrane (as shown in FIGS. 1-3). In some forms, the container includes a base or coupling base 102 having snap features 109,110 formed thereon that are configured to couple with another container, referred to as a negative pressure container 11, that includes an opening having snap features 112,111 formed around the opening that cooperate with the snap features of the base to couple the negative pressure container 11 to the base 107. The base 102 may be an annular structure 107 surrounding a planar surface 106, and the opening and perimeter of the vacuum container may be configured to correspond to the planar surface of the base such that when the vacuum container and base are connected, the opening and perimeter of the vacuum container can be engaged with the planar surface and the opening of the vacuum container and the underside of the membrane contact to form a gas-tight seal. Thus, when the negative pressure container contains liquid, such as air or liquid on the lower surface of the membrane, and the liquid falls with gravity, the air pressure on the lower surface of the membrane and the upper surface of the falling liquid falls, creating a negative pressure that drives the liquid on the membrane downward through the membrane. Of course, a vacuum pump can be connected to the lower opening of the negative pressure container, so that negative pressure can be formed to drive liquid on the membrane to flow to the lower membrane through the membrane hole.
Fig. 4-5 are schematic views of the structure in which the film-carrying container 10 is fixedly arranged on the support plate, and fig. 7 is an exploded perspective view of the structure. With such a structure, separation of a specific substance in a sample on the membrane, such as tumor cells and the like, can be achieved.
Example 1: effect of different frequencies on cell separation throughput and injury。
300 million human lymphoblasts (T2) were in 5mLPBS buffer. The filter bowl in the apparatus was equipped with a Parylene (Parylene) filter membrane (not easily deformable) having 100 ten thousand hexagonal micropore arrays with 6.9 μm inscribed circle diameter, with a membrane thickness of 10 μm. The number of intercepted cells on the membrane was estimated to be 3 times the number of micropores, and the system described in FIGS. 4-6 (in the absence of negative pressure vessel 11) was used, after vibration was turned on, 5mL of the above T2 cell suspension was added to the filter bowl for separation, and the total filtration time was measured to determine the separation flux. Under the same experimental conditions, 5ml of LPBS suspension containing 300 ten thousand of human chronic myelogenous leukemia cells (K562) and human acute T lymphocytes (Jurket) are respectively adopted as samples to be separated, and the statistical separation flux is shown in the following table.
Note: statistics of cell damage phenomena disruption was observed after DAPI fluorescent staining of the cells on the membrane after separation, and nuclear scattering occurred in part.
The film-carrying container of the present invention is fixed on a vortex instrument by adopting a traditional vortex (vortex instrument) mode, and the results are as follows:
through our experiments, under the condition of the same vibration frequency of 50HZ, the membrane separation is carried out on the same sample by adopting a vortex mode, the survival rate of target cells is only less than 50%, and most of the cells are separated but damaged to different degrees.
With respect to the first example, only the support plate (which is placed directly on the operating table), without the spring, has the following separating effect:
from the above experimental data, it can be seen that as the frequency of vibration increases, the separation flux increases and the efficiency increases, but as the frequency increases, different cells have the ability to withstand different impairments. T2 and K562 samples, cells remained intact and were rarely damaged when the frequency was 333 Hz. However, in the case of human acute T lymphocytes, target cells isolated under high frequency vibration are seriously damaged. This means that the system of the present invention needs to specify what type of target cell is when confirming the cell to be analyzed, and then select an appropriate frequency for vibration according to the target cell. If the spring according to the invention is not selected but only used with a support plate, the flux of detachment is reduced at increased vibration frequencies, causing different degrees of damage to the cells, which is seen as an important role of the support plate with the spring, especially at high frequencies, which plays a particularly important role for the damage of the cells.
As described above, in each micro-pore, due to the excessive cell clogging, a small amount of liquid will flow into the membrane under the action of the pressure difference between the capillary action and the membrane (or under the action of the pressure difference or gravity), but the precise separation function of the membrane cannot be performed, and the concentration efficiency will be low.
When using vibrational power, the support plate is subjected to the vibrational waves from the shimming surfaces of the spring system and transmits such vibrations to the membrane, causing the desired cells (tumors, T cells, human lymphoblasts (T2)) and other components on the membrane to also be subjected to the vibrational waves, to move away from the surface of the membrane, to rearrange randomly with the overlying and surrounding components, to be screened again by the microwells with other undesired components, so that the undesired components flow through the microwells to the other side of the membrane while the desired cells remain on the membrane. The invention has springs, and the entire vibration table is placed on the spring assembly. The spring assembly plays two roles, one is to isolate the mechanical connection between the vibration system and the outside, and avoid energy loss; another more critical function is to convert the vibration generated by the point-like vibration source into the planar vibration source parallel to the microporous array membrane as much as possible through the spring system, so that the vibration can be transmitted to the separation system and the membrane surface more efficiently and uniformly. Therefore, the whole spring system has the functions of vibration isolation and vibration converter, not only reduces unnecessary vibration energy loss, but also can properly inhibit macroscopic displacement in the horizontal direction, and better and uniformly apply energy on the membrane plane, thereby overcoming the defect that the inner wall of the filter cavity possibly generates overlarge influence on liquid and even generates macroscopic vortex under the condition of high frequency, such as accompanying high amplitude, so that local target cells are damaged. Generally, it is desired to generate a relatively low amplitude at a high frequency to achieve separation and to ensure maximum cell activity. Therefore, the designed spring system can improve the efficiency of the vibration filtering system, simultaneously avoid generating large macroscopically inhomogeneous local high-shear force flow fields, ensure the efficiency of the cell separation system and simultaneously avoid the damage of cells caused by local high-shear force.
Example 2 of implementation: influence of vibration frequency on separation flux
100 ten thousand T2 cells (total number) were put in 5ml PBS buffer, and in the apparatus shown in FIGS. 5 to 6, a Polycarbonate (PC) micropore array filter having 40 ten thousand round holes with a diameter of 8 μm was provided, the number of cells was 2 to 3 times the number of micropores (negative pressure was applied to the negative pressure vessel 11), and the total filtration time was measured while shaking at a negative pressure of 10kPa to determine the separation flux.
Example 3:effect of vibration frequency on cell rejection
300 million Chinese hamster ovary cells (CHO cells, cell diameter 14-20 μm) (total number) in 5ml PBS buffer, in the apparatus of the figure, a filter membrane of Parylene (Parylene) microporous array having 100 ten thousand 6, 8.6 and 10.4 μm inner circle diameter regular hexagonal holes, respectively, was provided, the number of cells was 3-4 times the number of micropores, and the number of cells before and after filtration was compared while shaking. The cell removal effect was good at each frequency, but the index was also related to the size of the microwells themselves. When the cell is close to the pore diameter, the vibration may cause the cell which is originally trapped on the membrane to be easier to deform and pass through the micropore, the CHO cell filtration of the regular hexagon micropore array membranes with the inscribed circle diameters of 8.6 mu m and 10.4 mu m under the assistance of high-frequency vibration has a part of cells with small size and strong deformability to pass through the filter membrane, and the cell sizes in the filtrate are measured to find that the cell diameters are respectively 14.7 +/-0.3 mu m and 15.4 +/-0.1 mu m. Whereas if 10 micron polystyrene microspheres (PS material) without deformability were used, 100% was retained by separation under the same conditions with a Parylene (Parylene) microporous array filter membrane having 8.6 μm inner circular diameter regular hexagonal holes under 120Hz shaking. Therefore, the cell via hole larger than the size of the micropore under the vibration condition is mainly achieved by self deformation, and is not caused by deformation and pore size distortion of the flexible micropore film under the vibration condition. Thus, the vibration system is proved to guarantee the separation precision of the ultra-low pressure micropore array membrane.
300 ten thousand K562 cells (total number) were placed in 5ml PBS buffer, and in the apparatus of FIGS. 5-6, a Polyimide (PI) micropore array filter membrane having 100 ten thousand square holes with an 8.0 μm inscribed circle diameter was prepared, the number of cells was 3-4 times the number of micropores, filtration was performed while shaking, the membrane and cells on the membrane were placed in a release solution after filtration, and the ratio of recovered cells to original cells was counted.
Example 5:influence of vibration on cell size screening of microporous array membranes with different apertures
300 million CHO cells (total) in 5ml PBS buffer, in the apparatus of FIGS. 5-6, regular hexagonal Parylene microwell array filters with 100 million different well diameters, 3-4 times the number of microwells, filtered while shaking at 70Hz, and the average size (diameter) of the cells trapped on the membranes of different microwell sizes was counted.
The vibration changes the via function of the microporous membrane separation to some extent, and the pressed via occurs when the cell diameter is close to (equal or substantially equal to) the micropore diameter. Therefore, when the pore size parameter is selected under the vibration condition, it is necessary to select a membrane smaller than the pore size of the objective cell. This may be for those micropores
And the application of vibration to the recovery of the separated cells is also related to the pore size of the membrane of the microwell array. At a vibration frequency of 50Hz, when 200 million Chinese hamster ovary cells (CHO cells with a cell diameter of 14-20 μm) are placed in a regular hexagonal microporous membrane with a micropore diagonal of 4, 6 and 8 μm, few cells are stuck into the microporous membrane, a small amount of cells are adhered to the surface of the microporous membrane (FIG. 8A and FIG. 8B), and the diameter of the cells is larger than the pore diameter of the microporous membrane. However, in the 10 and 12 μm microporous membranes, after the separation by vibration, there is a situation that the residual cells on the membrane surface are stuck into the micropores (fig. 8D and fig. 8E), because the vibration will change the pore size of the membrane itself, and generally will increase a little, so the target cells are stuck in the pores, and the sticking in the pores is not convenient to separate the cells from the membrane, and in addition, the empty blockage of the membrane will be caused, and the cells are stuck in the pores of the membrane, and the separation cannot be effectively realized. In contrast, in the control group, CHO cells were filtered without shaking, and the cells remained on the membrane, which made it difficult to easily release and recover the cells, and also did not exhibit the separation effect (experimental data is omitted).
Finally, it should be noted that: the above-mentioned embodiments are only specific embodiments of the present invention, which are used for illustrating the technical solutions of the present invention and not for limiting the same, and the protection scope of the present invention is not limited thereto, although the present invention is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: any person skilled in the art can modify or easily conceive the technical solutions described in the foregoing embodiments or equivalent substitutes for some technical features within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the embodiments of the present invention, and they should be construed as being included therein. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
The invention shown and described herein may be practiced in the absence of any element or elements, limitation or limitations, which is specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It should therefore be understood that although the present invention has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The contents of the articles, patents, patent applications, and all other documents and electronically available information described or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.
Claims (11)
1. A separation system for target substances in a biological fluid sample, the system comprising: a microwell array membrane with which a biological fluid sample is received or contacted; a vibration power source component capable of generating vibration, and a transmission component for transmitting the vibration generated by the vibration power source to the micropore array membrane, thereby separating the target substance in the biological fluid through the membrane.
2. The separation system according to claim 1, wherein the microporous array membrane comprises 500-50000000 micropores per square millimeter in unit area, the diameter of the micropores is 0.1-50 microns, the thickness of the membrane is 1-100 microns, and the porosity is 5% -90%.
3. The separation system according to claim 1, wherein the driving member comprises a support plate for fixing the membrane of the micro well array and a spring for suspending the support plate while converting and homogenizing energy of the vibration source fixed to the support plate.
4. The separation system according to claim 1 wherein the source of vibratory drive power comprises various mechanical vibration generators such as a coreless vibratory motor and/or an eccentric dc motor; the ultrasonic generator based on the transducer chip and/or the pneumatic vibration device are one or more of.
5. The separation system according to claim 3 wherein the spring is disposed below the support plate for supporting the support plate so that the support plate stands supported by the spring.
6. The separation system according to claim 1 wherein the membrane is disposed in a membrane carrier container, the container including the membrane and a chamber for receiving the sample, such that the liquid sample in the chamber is in contact with the membrane.
7. The separation system according to claim 6 wherein the support plate includes structure for mounting the membrane carrier, whereby the membrane carrier is removably secured to the support plate.
8. The separation system according to claim 6 wherein said container includes an ultra low negative pressure element beneath the membrane for applying a negative pressure to the membrane.
9. A separation system as claimed in claim 8, wherein the sub-atmospheric pressure element comprises a lower chamber having an opening at one end which engages the base of the membrane carrier so that the membrane is sealed from the opening and sub-atmospheric pressure can be established between the lower chamber and the membrane.
10. The separation system according to claim 1, wherein the target substance in the biological fluid sample is one or more of tumor cells, abnormal circulating cells, exfoliated tumor cells, cell lines capable of producing antibodies or antigens, lymphocytes or stem cells of autologous or allogeneic origin, adipocytes, macrophages, fungal spores, bacteria, viruses, and extracellular vesicles.
11. The separation system according to one of claims 1 to 10, wherein the frequency of the vibrational power is 8 to 333 hz when the target substance size is above 160% of the pore equivalent diameter size; when the size of the target substance is less than or equal to 160% of the diameter of the equivalent circular hole of the micropore, the frequency of the vibration power is 8-120 Hz.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110861683.XA CN113834799A (en) | 2021-07-29 | 2021-07-29 | Separation system for target substance in biological liquid sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110861683.XA CN113834799A (en) | 2021-07-29 | 2021-07-29 | Separation system for target substance in biological liquid sample |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113834799A true CN113834799A (en) | 2021-12-24 |
Family
ID=78963018
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110861683.XA Pending CN113834799A (en) | 2021-07-29 | 2021-07-29 | Separation system for target substance in biological liquid sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113834799A (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56154988A (en) * | 1980-05-02 | 1981-11-30 | Olympus Optical Co Ltd | Releasing method of cultured cells and device therefor |
| US20150004702A1 (en) * | 2011-08-29 | 2015-01-01 | Stempeutics Research Private Limited | System for isolating stromal vascular fraction (svf) cells from the adipose tissue and a method thereof |
| US20170121664A1 (en) * | 2014-03-25 | 2017-05-04 | Tokyo Electron Limited | Cell separation device and cell separation method |
| CN207446383U (en) * | 2017-07-03 | 2018-06-05 | 南京工程学院 | Simultaneously the side-mounted double-mass vibrating of coil spring is not ground convexity |
| JP2019030260A (en) * | 2017-08-08 | 2019-02-28 | 学校法人慶應義塾 | Cell production method and cell production device |
| CN211111991U (en) * | 2019-11-19 | 2020-07-28 | 吉林省拓华生物科技有限公司 | Tissue source cell filter equipment |
| CN211436929U (en) * | 2019-11-05 | 2020-09-08 | 河南莲花酶工程有限公司 | Phytase finished product packing is with vibration screening device |
| CN213140466U (en) * | 2020-08-15 | 2021-05-07 | 河北飞龙行达生物科技有限公司 | Feed production is with pelletization feeder |
| CN112812945A (en) * | 2021-03-03 | 2021-05-18 | 北京大学第一医院 | Malignant pleural effusion tumor cell separation device and separation method |
| CN112915791A (en) * | 2021-02-09 | 2021-06-08 | 杭州佰迈医疗科技有限公司 | Ultra-low pressure membrane separation system and separation method |
| CN214347824U (en) * | 2021-01-27 | 2021-10-08 | 成都中合鑫建材有限公司 | Jarring formula sieving machine |
-
2021
- 2021-07-29 CN CN202110861683.XA patent/CN113834799A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56154988A (en) * | 1980-05-02 | 1981-11-30 | Olympus Optical Co Ltd | Releasing method of cultured cells and device therefor |
| US20150004702A1 (en) * | 2011-08-29 | 2015-01-01 | Stempeutics Research Private Limited | System for isolating stromal vascular fraction (svf) cells from the adipose tissue and a method thereof |
| US20170121664A1 (en) * | 2014-03-25 | 2017-05-04 | Tokyo Electron Limited | Cell separation device and cell separation method |
| CN207446383U (en) * | 2017-07-03 | 2018-06-05 | 南京工程学院 | Simultaneously the side-mounted double-mass vibrating of coil spring is not ground convexity |
| JP2019030260A (en) * | 2017-08-08 | 2019-02-28 | 学校法人慶應義塾 | Cell production method and cell production device |
| CN211436929U (en) * | 2019-11-05 | 2020-09-08 | 河南莲花酶工程有限公司 | Phytase finished product packing is with vibration screening device |
| CN211111991U (en) * | 2019-11-19 | 2020-07-28 | 吉林省拓华生物科技有限公司 | Tissue source cell filter equipment |
| CN213140466U (en) * | 2020-08-15 | 2021-05-07 | 河北飞龙行达生物科技有限公司 | Feed production is with pelletization feeder |
| CN214347824U (en) * | 2021-01-27 | 2021-10-08 | 成都中合鑫建材有限公司 | Jarring formula sieving machine |
| CN112915791A (en) * | 2021-02-09 | 2021-06-08 | 杭州佰迈医疗科技有限公司 | Ultra-low pressure membrane separation system and separation method |
| CN112812945A (en) * | 2021-03-03 | 2021-05-18 | 北京大学第一医院 | Malignant pleural effusion tumor cell separation device and separation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200399583A1 (en) | Acoustic perfusion devices | |
| US10953436B2 (en) | Acoustophoretic device with piezoelectric transducer array | |
| US9783775B2 (en) | Bioreactor using acoustic standing waves | |
| JP5827971B2 (en) | Filter cleaning apparatus and method with ultrasonic, backwash and filter motion during biological sample filtration | |
| AU2013249071B2 (en) | Acoustophoretic separation of lipid particles from red blood cells | |
| US9457302B2 (en) | Acoustophoretic device with piezoelectric transducer array | |
| US9623348B2 (en) | Reflector for an acoustophoretic device | |
| US8936160B2 (en) | Nozzle device and nozzle for atomisation and/or filtration and methods for using the same | |
| US20180010085A1 (en) | Acoustic perfusion devices | |
| US20170166860A1 (en) | Methods and apparatus for particle aggregation using acoustic standing waves | |
| WO1998017373A1 (en) | Method and apparatus for removing particles from suspension | |
| US20170217794A1 (en) | Transducer and reflector configurations for an acoustophoretic device | |
| JP2014003977A (en) | Adult stem cell isolation unit | |
| US10710006B2 (en) | Piezoelectric transducer for generation of an acoustic standing wave | |
| CN108136283B (en) | Large-scale acoustic separation device | |
| US20170218323A1 (en) | Acoustic separation of t cells | |
| CN113834799A (en) | Separation system for target substance in biological liquid sample | |
| WO2017132694A1 (en) | Acoustic perfusion devices | |
| WO2021054001A1 (en) | Filtration collection device | |
| US20170267991A1 (en) | Acoustophoretic clarification of particle-laden non-flowing fluids | |
| JP2022528345A (en) | Devices and methods for separating, filtering, and / or concentrating microparticles and / or nanoparticles. | |
| KR20180071254A (en) | Multipurpose Acoustic Float Capture Machine | |
| US20180264482A1 (en) | Separation of nanoparticles via acoustofluidic flow relocation | |
| EP4090732A1 (en) | Enhanced acoustic particle processing with seeding particles | |
| WO2017193085A1 (en) | Acoustophoretic clarification of particle-laden non-flowing fluids |
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 |