WO2020001662A1 - 稀有细胞捕获系统及其应用 - Google Patents

稀有细胞捕获系统及其应用 Download PDF

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Publication number
WO2020001662A1
WO2020001662A1 PCT/CN2019/094629 CN2019094629W WO2020001662A1 WO 2020001662 A1 WO2020001662 A1 WO 2020001662A1 CN 2019094629 W CN2019094629 W CN 2019094629W WO 2020001662 A1 WO2020001662 A1 WO 2020001662A1
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Prior art keywords
fluid
blood
capture
component
component capture
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PCT/CN2019/094629
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English (en)
French (fr)
Inventor
石虎兵
李燕雏
徐琪
孔祥菊
王学焱
韩赫文
Original Assignee
成都普瑞康生物科技有限公司
四川大学
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Priority to EP19826242.0A priority Critical patent/EP3816274B1/en
Priority to US17/255,381 priority patent/US12071611B2/en
Publication of WO2020001662A1 publication Critical patent/WO2020001662A1/zh

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    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00—General characteristics of the apparatus
    • A61M2205/02—General characteristics of the apparatus characterised by a particular materials
    • A61M2205/0244—Micromachined materials, e.g. made from silicon wafers, microelectromechanical systems [MEMS] or comprising nanotechnology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention relates to the detection of biological and pathological body fluids, in particular to a system for separating rare components from a biological body fluid sample and its application, and further relates to a system for separating rare cells from a whole blood cell population and its application, in particular to a system Large-capacity, online, low-hemolytic system for collecting circulating tumor cells from blood and its application.
  • Rare cells refer to some atypical cells in biological fluid samples (including blood, pleural fluid, ascites, urine, cerebrospinal fluid, etc.). Studies have shown that the collection of rare cells and the use of them to complete NGS analysis have important guiding significance for finding potential therapeutic mechanisms, pathological mechanisms and targeted drug development of the disease.
  • the research methods for detecting rare cells in blood mainly include flow sorting technology, morphological separation method, density gradient centrifugation method, membrane filtration method and immunomagnetic separation technology.
  • BD FACSAris can achieve high-speed cell sorting. The transient laser will damage the sorted cells, and the cell viability will be damaged after the sorting activity.
  • Circulating tumor cells CTCs refer to tumor cells that enter human peripheral blood.
  • CTCs in peripheral blood of cancer patients are very small, generally about 10 7 orders of magnitude CTCs in blood cells are only single digits (2-10 per ml), but CTCs are a very important liquid biopsy, a prognosis and Inter-treatment follow-up tool. Because the number of circulating tumor cells is extremely scarce, the accuracy and specificity of their detection are very high, and further analysis is more difficult. Therefore, it is urgent to develop efficient, high-throughput, fast circulating tumor cells from blood samples. Separated portable methods and tools.
  • Circulating tumor cells in human peripheral blood are tumor cells (CTCs) or cell clusters (CTM) that have been spread by the tumor lesions into the peripheral blood circulation. Surviving CTCs or CTM leave the blood circulation and enter the local microenvironment of secondary organs. Under the action of growth factors, proliferation and growth can eventually form metastases. Circulating tumor cells are an important source of tumor blood tract metastases, and distant metastasis is one of the direct causes of tumor treatment failure, recurrence and death.
  • CTCs the prerequisite for clinical diagnosis or laboratory analysis of CTC is to obtain sufficient CTC cells. Since in the peripheral blood CTCs per 10 6 to 10 7 mononuclear cells contain only a CTCs, therefore CTCs sensitivity and specificity of detection are made high demands. At present, the detection schemes of various CTCs mainly include CTCs separation and enrichment systems, and CTCs detection and identification systems.
  • the CellSearch TM system is the most representative CTCs separation and collection system. It has been approved by the US FDA for the detection of circulating tumor cells in metastatic breast cancer.
  • the CellSearch TM system uses magnetic particles labeled with antibodies against EpCAM to circulate tumors in the blood. Cell capture, but this system only detects 10ml of blood drawn. Compared with circulating blood, more potential circulating tumor cells have not been detected, and there is a selective offset of the sample.
  • the sample detected by this method is already in vitro. After being left for several hours, the cells are in a state of hypoxia or even hypoxia. At this time, the captured circulating tumor cells have lost their viability, and it is difficult to analyze signal pathways and functionalities, and it is impossible to perform in vitro culture, single-cell sequencing and other tasks.
  • microfluidic cell immune chip is to detect CTCs through a micro-fluid device.
  • the detection method has the characteristics of high specificity, sensitivity and repeatability.
  • the captured CTCs have cell activity and can be Isolation and use in cell culture and various other downstream technology researches are a new and efficient method for the clinical application value of CTCs.
  • the volume of blood samples detected by ordinary microfluidic cell immunochips is small, and there are still defects such as selective shift of samples.
  • CTCs The identification and characterization of CTCs provides opportunities for research, monitoring, and ultimately changing the transfer process.
  • a major limitation of most existing microfluidic systems is that device efficiency is limited by the shear stresses the device exerts on blood cells and CTCs. Too much shear stress will destroy blood cells and CTCs.
  • CTC When CTC ruptures, the target isolate (CTC itself) is lost, and the internal group of CTC is dispersed into the fluid.
  • blood cells mainly white blood cells
  • the intracellular material released further complicates non-specific interactions between species in the blood, making high-purity CTC extraction more difficult.
  • Some existing devices have attempted to avoid damage to cells, including blood cells and CTC, by using low-channel microfluidic devices to reduce shear stress.
  • low channels cause a reduction in the volume of fluid flowing through the channels, which in turn results in very low flow rates.
  • Such a device is not sufficient to quickly process large amounts of blood to make it practical and useful.
  • rapid processing of large volumes (7-30 mL) of blood is a prerequisite for high-yield separation of intact CTCs.
  • the whole blood can not be screened and analyzed. There are problems such as small blood collection samples, insufficient collection of CTCs, and selective sample migration.
  • Connecting the existing external CTCs collection and separation device online to the blood circulation in the body may cause damage to the cells in the blood, and there is a risk of hemolysis.
  • the device will cause cell fragments after the hemolysis to promote blood clotting and blood quality degradation. May cause potential clinical risks. Therefore, there is a need for a system that can access the blood circulation in the body, collect CTCs online, and reduce the risk of hemolysis, so that the treated blood meets the blood transfusion standard (1, no severe hemolysis; 2, in vitro time does not exceed 6 hours; 3, cannot Use a liquid that can hemolyze red blood cells to clean or process the blood), an easy-to-use, online, large-capacity, low hemolytic circulating tumor cell collection system.
  • the inventor of the present invention has provided a tumor cell collection system satisfying the above-mentioned standards through creative work.
  • the above system has the following advantages: 1. While ensuring CTC capture efficiency, it guarantees hemodynamics and the state of cells in the blood, and meets the blood transfusion standard; 2. It uses a microfluidic chip to give a small range of anticoagulants, and the whole body Compared with anticoagulation, the total amount of anticoagulant is reduced and the risk of systemic anticoagulation is reduced; 3.
  • the system of the present invention can not only capture rare cells on-line extracorporeal circulation, but also realize extracorporeal circulation of fluids offline, And improve capture efficiency. 8.
  • the system of the present invention can be used to capture not only rare cells in blood, but also various cells or cell components in other biological fluids.
  • the object of the present invention is to provide an online large-capacity circulation rare cell capture system and its application.
  • the present invention provides a fluid component capture system, the system comprising a fluid pipeline device, a circulating power plant device, a component capture device, and an optional anticoagulant release device; wherein the circulating power plant device
  • the component capture device is connected in series to the fluid circulation system through a fluid pipeline device to form an extracorporeal fluid circulation path; wherein the component capture device includes a microfluidic chip or chipset, and the microfluidic chip includes a spoiler array inside .
  • the present invention provides a fluid component capture system, the system comprising a fluid pipeline device, a circulating power plant device, a component capture device, and an optional anticoagulant release device; wherein the circulating power plant device
  • the component capture device is connected in series to the fluid circulation system through a fluid pipeline device to form an extracorporeal fluid circulation path.
  • the component capture device includes a microfluidic chip or chipset, and the internal corners of the microfluidic chip are smoothly transitioned. Surface.
  • the present invention provides a fluid component capture system, the system comprising a fluid pipeline device, a circulating power plant device, a component capture device, and an optional anticoagulant release device; wherein the circulating power plant device And the component capture device are connected in series to the fluid circulation system through a fluid pipeline device to form an extracorporeal fluid circulation path; wherein the component capture device includes a microfluidic chip or chipset; wherein the circulation power device device includes a simulated heart squeeze Bionic pump.
  • the invention provides a microfluidic chip.
  • the present invention provides a circulation power device that includes a bionic pump that mimics the compression of a heart to provide power for extracorporeal fluid circulation.
  • the component is a circulating rare cell, preferably a circulating tumor cell.
  • the fluid pipeline equipment further comprises a bypass pipeline in parallel with the component capture equipment
  • the bypass pipeline is provided with a flow limiting valve or a speed limiting valve that can adjust the flow rate, preferably
  • the restriction valve is a three-way valve.
  • an anticoagulant release device is provided between the circulating power plant equipment and the component capture device, said anticoagulant release device comprising an anticoagulant slow-release device, and optionally, said anticoagulant
  • the coagulant slow-release device is connected to the fluid pipeline through a three-way valve.
  • the microfluidic chip main body includes a substrate layer and a cover sheet layer arranged in order from bottom to top, and a component capture chamber is provided between the substrate layer and the cover sheet layer, the The cover sheet layer is provided with a fluid inlet and a fluid outlet, which are in communication with the component capture chamber.
  • the component capture chamber is divided into a buffer zone and a swim lane.
  • the swim lane is provided with an array of spoiler columns along the flow direction.
  • the corner surfaces of the component capture chamber are all smoothly curved surfaces. Those skilled in the art can understand that the corners include both concave corners, such as the connection between the upper and lower walls of the component capture chamber and the side wall.
  • the shunt block or the spoiler At the connection between the shunt block or the spoiler and the upper and lower walls, it also includes convex corners, such as the angle of the shunt block or the spoiler protruding outward in the direction of fluid flow.
  • the buffer zone is near the entrance and the exit and is the junction of the swim lanes, which has the function of reducing the flow velocity and buffering.
  • each swim lane contains 5-500 spoilers, preferably 10-200, further preferably 15 -100, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, most preferably 17; optionally, the spoilers are parallel in the swim lane at a specific distance Arranged, the pitch is 0.1-5, preferably 0.5-1 diameter (long diameter or short diameter) of the spoiler.
  • the two ends of the shunt block respectively form the entrance and exit portions of the swim lane, and the corners of the entrance and exit surfaces are treated with anti-hemolysis treatment to form a smooth transition curve.
  • the cross section of the entrance and exit may be a circular arc. Shape, round shape, rounded rectangle, rounded trapezoid, etc.
  • a corner surface of the spoiler in a flow direction is a curved surface with a smooth transition.
  • the inner surface of the component capture chamber and the surface of the spoiler are loaded with a capture ligand that binds to a target component in a fluid
  • the capture ligand is streptavidin- Biotin-EPCAM complex.
  • the material of the substrate or cover sheet is selected from silicon, glass, siliconized glass, PDMS, or is selected from polypropylene, cycloolefin copolymer, cycloolefin polymer, polymethyl methacrylate, and One or two or more types of high molecular polymer materials in polycarbonate.
  • the circulation power device equipment includes a bionic pump that mimics the compression of the heart to provide power for extracorporeal fluid circulation
  • the bionic pump includes an elastic bladder with inlets and outlets at both ends; a torsion piece, It is sleeved on the outer periphery of the elastic liner;
  • a drive assembly includes a power output member, and the power output member is fixedly connected to the torsion member, so that the torsion member can be driven to rotate when the power output member is rotated
  • the elastic liner is squeezed or released radially along the elastic liner.
  • a one-way valve is connected at the inlet and outlet of the elastic liner to simulate the function of a heart valve and realize a one-way flow of blood.
  • the torsion member includes a spirally wound torsion body, a first end of the torsion body is fixedly disposed, and a second end of the torsion body is fixedly connected to the power output member;
  • the torsion member includes a torsion member main body, the torsion member main body includes a plurality of rod members that are surrounded in parallel to form a columnar structure, a first end of the torsion member main body is fixedly disposed, and a second end of the torsion member main body It is fixedly connected with the power output member.
  • the torsion member further includes a first protective layer covered on an inner periphery of the torsion member body; and / or the torsion member further includes a second protective layer covered on an outer periphery of the torsion member body.
  • the protective layer is not limited to any one of the preceding aspects.
  • the power output member in the circulating power plant equipment includes a motor, a speed governor, and a reversing mechanism
  • the speed governor is a full-automatic forward and reverse DC motor speed governor, preferably
  • the reversing mechanism is a bevel gear set.
  • the interval and speed of the forward and reverse rotation of the motor are controlled by a fully automatic forward and reverse DC motor speed controller, and the setting parameters include: the interval between the forward and reverse rotation of the motor is 0.5 to 1 second, The motor speed is 30-60 times / minute.
  • the present invention relates to a power device for a blood cell collection system, characterized in that the power device includes:
  • a torsion member sleeved on the periphery of the elastic liner
  • the driving assembly includes a power output member, and the power output member is fixedly connected to the torsion member, so that when the power output member rotates, the torsion member can be driven to twist to squeeze or release the elastic liner.
  • the torsion member includes a torsion member main body wound spirally, a first end of the torsion member main body is fixedly disposed, a second end of the torsion member main body and the power output member Fixed connection; or
  • the torsion member includes a torsion member main body, and the torsion member main body includes a plurality of rod members that are surrounded in parallel to form a columnar structure.
  • the power output member is fixedly connected.
  • the torsion member further includes a first protective layer covered on an inner periphery of the torsion member body; and / or the torsion member further includes a first protective layer covered on the torsion member body. Second protective layer.
  • the power output member includes an end surface perpendicular to the power output axis, and the second end of the torsion body is fixedly connected to the end surface.
  • the power output member includes a motor and a reversing mechanism, the reversing mechanism being connected to the motor for changing a power output direction of the motor.
  • the reversing mechanism is a bevel gear set
  • the motor is connected to a driving wheel of the bevel gear set
  • the end surface is located on a driven wheel of the bevel gear set.
  • the power output member includes a motor and a flange connected to an output shaft of the motor, and the end surface is located on the flange.
  • the driving component further comprises a controller, the controller is electrically connected to the motor to control the motor to perform a forward and reverse operation under a set parameter.
  • a method of capturing rare cells in blood comprising using the fluid component capture system of any of the preceding aspects to capture rare cells in blood.
  • a method according to the preceding aspect comprising the following steps:
  • the blood from the blood collection device is passed through the circulation power device in the fluid pipeline, and then enters the microfluidic chip for cell capture.
  • the blood collection device may be a blood collection needle or a blood container
  • the blood transfusion device may be a blood transfer needle or a blood container that is the same as or different from the blood collection device.
  • FIG. 1 Schematic diagram of fluid component capture system, where 11 is an elastic liner, 111 is a power unit inlet, 112 is a power unit outlet, 2 is a fluid pipeline, 3 is a cell capture device, 4 is a human body, and 5 is an anticoagulant release device .
  • Figure 2 Schematic diagram of circulating power plant equipment
  • Figure 2A is an embodiment of the torsion piece
  • Figure 2B is a schematic diagram of one embodiment of circulating power plant equipment, where 1 is a circulating power plant, 2 is a fluid pipeline, and 11 is an elastic liner
  • 12 is the main body of the torsion member
  • 13 is the flange
  • 14 is the motor
  • 15 is the check valve
  • 16 is the second protective layer
  • 112 is the power unit outlet
  • Figure 2C is a schematic diagram of another embodiment of the circulating power unit equipment , Where 12 is the main body of the torsion, and 13 is the flange.
  • FIG. 3 is a schematic diagram of a microfluidic chip and its connection
  • FIG. 3A is a schematic diagram of an embodiment of the microfluidic chip
  • FIG. 3B is a schematic diagram of a microfluidic chipset and a bypass pipe connection.
  • rare cells refer to some atypical cells in biological fluid samples (including blood, pleural fluid, ascites, urine, cerebrospinal fluid, etc.).
  • examples of rare cells include, but are not limited to, circulating tumor cells (CTC), circulating endothelial cells (CEC), circulating multiple myeloma cells (CMMC), and circulating melanoma cells (CMC).
  • CTC circulating tumor cells
  • CEC circulating endothelial cells
  • CMMC circulating multiple myeloma cells
  • CMC circulating melanoma cells
  • Preferred rare cells are CTC and CEC, and particularly preferred rare cells are CTC.
  • CTC cancer cells detected in the circulating blood of a subject.
  • Analyte refers to a molecule or component in a fluid that is the target of a method of detection, separation, concentration, or extraction.
  • exemplary analytes include cells, viruses, nucleic acids, proteins, carbohydrates, and small organic molecules.
  • Blood component refers to any component of whole blood, including host red blood cells, white blood cells, and platelets. Blood components also include plasma components such as proteins, lipids, nucleic acids, and carbohydrates, as well as any other cells, including rare cells, that may be present in the blood due to, for example, current or past pregnancy, organ transplantation, or infection.
  • Fluid or “biological fluid” is intended to include natural fluids (such as blood, lymph, cerebrospinal fluid, urine, cervical lavage fluid, saliva, and water samples), some of these fluids and fluids that have been introduced into cells (e.g., culture media And liquid tissue samples).
  • the term also includes lysates.
  • the "capture unit” or “capture ligand” may refer to a chemical sample for binding an analyte, or a component binding substance on the surface of a whole cell by which it is bound.
  • the capture unit may be a compound coupled to the surface or a material constituting the surface.
  • Typical capture units include antibodies, oligonucleotides or polypeptides, nucleic acids, other proteins, synthetic polymers, and carbohydrates.
  • a “channel” or “lane” refers to a gap through which fluid can flow.
  • the channel may be a capillary on a hydrophobic surface, a conduit, or a hydrophilic texture of a hydrophobic surface where an aqueous liquid may be confined.
  • a “component” of a cell refers to any component that can be isolated in a cell lysate.
  • Cell components can be organelles (e.g., nucleus, near nuclear compartment, nuclear membrane, mitochondria, chloroplast, or cell membrane), polymers or molecular complexes (e.g., lipids, polysaccharides, proteins (membrane, transmembrane, or cytoplasm), Nucleic acid (natural, therapeutic or pathogenic), viral particles or ribosomes) or other molecules (eg, hormones, ions, cofactors or drugs).
  • a “component” of a cell sample refers to the set of cell group molecules contained in the sample.
  • An "enriched sample” refers to a sample containing an analyte that has been processed to increase the relative content of the analyte relative to a sample that is usually present.
  • samples can be enriched by increasing the amount of target analyte by a factor of at least 10%, 25%, 50%, 75%, 100% or at least 10, 100, 1000, 10,000, 100,000, or 1,000,000 times.
  • a "rare amount" of cells refers to less than 100 cells / ml fluid, less than 10 cells / ml fluid, or even less than 1 cell / ml fluid.
  • the system includes a fluid pipeline device, a cell capture device, and a circulating power plant device.
  • the fluid pipeline device can be connected to the fluid circulation system to form In the extracorporeal fluid circulation pathway, the cell capture device is connected in series or in parallel with the fluid pipeline, so that the fluid can pass through the cell capture device, thereby capturing the cells contained in the fluid; the circulation power device equipment provides power for extracorporeal fluid circulation.
  • the fluid cell capture system further includes an anticoagulant release device, the anticoagulant release device is an anticoagulant slow-release device, the release device has a flow control function infusion pump, and the anticoagulant includes It is not limited to common anticoagulants such as EDTA, citric acid or heparin.
  • the fluid is blood.
  • the cells are circulating rare cells, and further preferably, the cells are circulating tumor cells.
  • the cell capture device includes a microfluidic chip or chipset.
  • the chip main body includes a base sheet layer and a cover sheet layer arranged in order from bottom to top, and a component capture chamber is provided between the base sheet layer and the cover sheet layer.
  • a fluid inlet and a fluid outlet are provided, which are in communication with the component capture chamber.
  • the component capture chamber is divided into a buffer zone and a swim lane.
  • the buffer zone is near the entrance and the exit.
  • the lane part includes one or more lanes, preferably divided into at least two lanes by a shunt block, and an array of spoiler columns is arranged along the flow direction in the lanes.
  • the two ends of the shunt block respectively form part of the entrance and exit of the swim lane, and the corner surface of the entrance and exit is treated with anti-hemolysis treatment to form a smooth transition surface.
  • the cross section of the entrance and exit may be arc-shaped, inverted Round, rounded rectangle, rounded trapezoid, etc.
  • the cross-section of the spoiler is streamlined, spindle-shaped, dumbbell-shaped, or similar to the shape where two water droplets end-to-end so that its surface in the direction of flow is a smooth transition surface, thereby reducing mechanical shear Damage to cells in a fluid by force.
  • the height of the spoiler array is equal to the internal height of the chip component capture chamber.
  • a spoiler is disposed on a side of the cover sheet close to the substrate.
  • the spoiler is disposed on the side of the substrate near the cover sheet.
  • each lane is 3mm-5mm
  • the height is 50-100 ⁇ m
  • the diameter of the spoilers is 0.1-5, preferably 0.5-1
  • the diameter of the spoiler array is 10-100 ⁇ m.
  • Each lane contains 50-500 spoilers to form a microarray.
  • the lanes are arranged in parallel.
  • the length of the substrate is 5-100 mm, preferably 20-80 mm, further preferably 30-60 mm, and most preferably 50 mm; the width is 5-50 mm, preferably 10-30 mm, and most preferably 20 mm.
  • the number of lanes is 1-20, preferably 4-15, further preferably 6-10, and most preferably 8.
  • the length of the lane is 5-100mm, preferably 20-40mm, most preferably 30mm; the width is 0.1-50mm, preferably 0.5-5mm, further preferably 1-3mm, most preferably 1.5mm; the height is 0.05 -0.5mm, preferably 0.05-0.1mm.
  • the material of the substrate or cover sheet is silicon, glass, siliconized glass, PDMS, etc., and may also be selected from polypropylene, cycloolefin copolymer, cycloolefin polymer, and polymethyl methacrylate. And polycarbonate one or two or more kinds of high molecular polymer materials.
  • the cell capture device includes a chip combiner, which is a medical-grade connection pipe and a card slot for placing a microfluidic chip and connecting multiple chips in parallel or in series to form a chipset.
  • a chip combiner which is a medical-grade connection pipe and a card slot for placing a microfluidic chip and connecting multiple chips in parallel or in series to form a chipset.
  • the inner surface of the component capture chamber and the surface of the spoiler are loaded with streptavidin, which is capable of interacting with epithelial cell adhesion molecule (EPCAM) -biotin
  • EPCAM epithelial cell adhesion molecule
  • the labeled biotin in the (Biotin) complex specifically binds, and the EPCAM-biotin complex can specifically bind to a unique antigen on the surface of circulating tumor cells, wherein streptavidin-biotin-EPCAM complex is streptavidin
  • the link to the biotin-biotin complex has the ability to compete for elution by high concentrations of biotin.
  • other capture ligands such as antigens, antibodies, protein A, protein G, lectin, etc. may also be coated on the inner surface of the component capture chamber and the surface of the spoiler.
  • the circulating power plant equipment includes an elastic liner with inlets and outlets at both ends; a torsion member sleeved on the outer periphery of the elastic liner; a drive assembly including a power output member, The power output member is fixedly connected to the torsion member, so that when the power output member is rotated, the torsion member can be driven to twist to squeeze or release the elastic liner.
  • the torsion member includes a spirally wound torsion member main body, a first end of the torsion member main body is fixedly disposed, and a second end of the torsion member main body is fixedly connected to the power output member; or
  • the torsion member includes a torsion member main body, and the torsion member main body includes a plurality of rod members that are surrounded in parallel to form a columnar structure. A first end of the torsion member main body is fixedly disposed, and a second end of the torsion member main body and The power output member is fixedly connected.
  • the torsion member further includes a first protection layer covered on the inner periphery of the torsion member body; and / or the torsion member further includes a second protection covered on the outer periphery of the torsion member body.
  • a first protection layer covered on the inner periphery of the torsion member body
  • a second protection covered on the outer periphery of the torsion member body.
  • the power output member includes an end surface perpendicular to the power output axis, and the second end of the torsion body is fixedly connected to the end surface.
  • the power output member includes a motor, a fully automatic forward and reverse DC motor speed controller, and a reversing mechanism, and the reversing mechanism is connected to the motor for changing the power output direction of the motor .
  • the reversing mechanism is a bevel gear set
  • the motor is connected to a driving wheel of the bevel gear set
  • the end surface is located on a driven wheel of the bevel gear set.
  • the power output member includes a motor and a flange connected to an output shaft of the motor, and the end surface is located on the flange.
  • the driving assembly further includes a controller, and the controller is electrically connected to the motor to control the motor to perform a forward and reverse operation under a set parameter.
  • a full-automatic forward and reverse DC motor speed controller is used to control the forward and reverse interval time and speed of the motor.
  • the setting parameters include: the interval between the forward and reverse rotation of the motor is 0.5 to 1 second, and the motor The rotation speed is 30-60 times / minute.
  • a protective layer is provided on the inner periphery of the main body of the torsion member, which can effectively increase the contact area of the main body of the torsion member with the elastic liner during torsion, thereby making the elastic liner more uniform in force and realizing the elastic liner. Evenly compresses or relaxes.
  • the torsion member is fixed on the end face of the flange or bevel gear, and then the motor output shaft is connected to the flange or bevel gear so that the axis of the power output shaft can coincide with the center line of the torsion member. Avoid the phenomenon of vibration caused by the offset between the connection point of the torsion member and the power output shaft and the shaft center of the output shaft, so that the power device of the system can achieve stable driving.
  • a one-way valve is connected at the inlet and the outlet of the elastic liner, simulating the function of a heart valve, realizing the one-way flow of blood, and avoiding the phenomenon of blood reflux during circulation to better protect Blood cells in the blood.
  • the torsion member and the elastic liner are detachable designs, which realizes the modularization and miniaturization of the device.
  • the elastic liner as a blood chamber is an enlarged,
  • the flexible device can effectively reduce the change of hemodynamic shear force during blood circulation, avoid the peristaltic pump from squeezing the red blood cells in the hose, and greatly reduce the risk of hemolysis.
  • the torsion piece By setting a torsion piece, the torsion piece is deformed to realize the squeezing and release of the elastic liner, and the motor is driven under the set parameters, that is, the motor is controlled by a fully automatic forward and reverse DC motor speed controller
  • the forward and reverse interval and rotation speed achieve the elastic inner tank to contract or relax with the same or similar motion pattern and rhythm as the heart beat.
  • the bionic drive of the power device While ensuring the efficiency of circulating tumor cell capture, the bionic drive of the power device is realized, making The power device can simulate the blood pumping mode of the heart, avoiding the damage of cells caused by excessive squeezing of the hose in the prior art, thereby ensuring the integrity of the cells in the transport process and reducing the impact of the blood collection system on the blood collection process. The destruction of blood cells greatly reduces the risk of hemolysis.
  • the anticoagulant release device is an anticoagulant slow-release device, preferably a miniaturized front-end anticoagulant slow-release device, and the front-end means to be placed before blood enters the microfluidic chip. Miniature anticoagulant slow release device to ensure the anticoagulation state in the microfluidic chip.
  • the slow-release device can be a conventional medical analgesia pump in the prior art.
  • the overall device is made of medical-grade plastic, which is portable and has the ability to release heparin sodium at a uniform speed, ensuring the anticoagulation in the environment of the microfluidic chipset.
  • the microfluidic chipset described in the present disclosure is connected to the system in a serial or parallel manner according to the actual blood collection demand. After the blood is guided through the blood vessel, it enters the circulation power system of the present disclosure to obtain extracorporeal circulation power, and is bifurcated into one or more parallel catheters at the distal end, which are respectively connected to the microfluidic chipset and the bypass, and enter the microfluidic. After the chipset, a flow limiting valve is set. See Figure 3 for connection.
  • the restriction valve is a three-way restriction valve.
  • the microfluidic chip or chipset is connected to the blood circulation system, and the anticoagulant slow-release device is connected in the form of a tee.
  • the anticoagulant slow-release device is an elastic automatic contraction pump, which shrinks and squeezes out the anticoagulant at a constant speed to ensure that the anticoagulant is pumped out at a constant speed.
  • a check valve is installed in the anticoagulant device to prevent microflow. Controlled blood circulation back into the anticoagulator; anticoagulant contains heparin sodium anticoagulant.
  • the anticoagulant slow-release device is set so that the anticoagulant pumping rate is a fixed value, and the output is adjusted according to the total volume of blood to be collected.
  • the pump body works and the anticoagulant is continuously pumped at a constant amount. .
  • the anticoagulant is mixed with the circulating blood, it passes through the microfluidic chip microchannel to contact the surface-loaded antibody, and the circulating tumor cell is captured from the blood sample by using the combination of the microchannel surface-loaded antibody and the circulating tumor cell surface-specific antigen. It was fixed on the surface of the microchannel and continuously collected for 1-10 hours.
  • the antibody and the circulating tumor cells captured by the streptavidin-biotin complex are released from the surface of the microchannel by cutting off the streptavidin-biotin complex, thereby obtaining high-purity circulating tumor cells, and after the end of the cycle collection
  • the microfluidic chip is washed with a cell washing solution, thereby achieving collection of the captured circulating tumor cells.
  • the cell washing solution is a buffer solution containing a high concentration of biotin protein, and a preservative can be added according to the needs of storage.
  • CD45 and CK8 / 18 fluorescent antibodies were used to stain the eluted CTC cells, and the CD45 (-) and CK8 / 18 (+) cell populations were identified as CTC cells by fluorescence microscope observation.
  • the low hemolytic property described in the present disclosure is verified by the measured red blood cell hemolysis after passing through the chip of the present disclosure after the anticoagulated blood detected by the “spectrophotometry” (FIG. 4).
  • the present invention has the following advantages and characteristics:
  • the existing system only analyzes a small amount of blood samples. Compared with the total blood volume of 4000ml in the whole body, the system sampling volume is too small, which may cause selective offset errors and false negative rates.
  • the sampling samples of the present invention can cover the whole body blood and solve the current CTC.
  • the problem that the collection scheme cannot scan and detect the whole body blood greatly reduces the system error caused by the selective shift of the sample; 2.
  • the present invention does not need to collect CTC Red blood cell lysis was performed before. On the contrary, it is necessary to protect red blood cells during the CTC collection process. 3.
  • the streptavidin-biotin complex combination of the present invention can be interrupted by the cell cleaning solution, which can pass the microfluidic-based
  • the non-invasive removal of circulating tumor cells captured by the control chip technology for further analysis or in vitro culture will better play the role of circulating tumor cells.
  • the entrances and exits of the microfluidic chipset and the entrances and exits of the connecting pipelines are designed as smooth transition curves to avoid the potential damage to the blood cells caused by the internal structure of the system; 5.
  • the spoiler column microarray increases the surface area in contact with blood. It also prevents mechanical damage to blood cells. Compared to existing CTC detection or blood cell removal devices, the spoiler chip designed by this patent is more suitable for online blood collection systems.
  • Blood collection is performed in the form of chipsets to ensure efficient CTC collection. And reduce the occurrence of blood hemolysis, and ensure the quality of blood after circulation. 6
  • the system of the present invention can not only capture rare cells on-line extracorporeal circulation, but also realize extracorporeal circulation of fluid offline, thereby improving the capture efficiency. 7.
  • the system of the present invention can be used to capture not only rare cells in blood, but also various cells or cell components in other biological fluids.
  • 100ul of streptavidin protein was injected into the chip with a syringe, and then the chip was placed in a wet box and coated at 37 ° C for 2 hours.
  • 100ul of streptavidin protein was injected into the chip with a syringe, and then the chip was placed in a wet box and coated at 37 ° C for 2 hours.
  • the power part of the device to the MCF7 breast cancer cell suspension system (simulated blood system, volume 100ml, 100 cells in the system). After the blood is guided out through a blood vessel, it enters the power device system (including a motor and a bionic pump) of the present invention to obtain extracorporeal circulation power, and then is connected to a blood circulation system composed of a microfluidic chipset.
  • the power device system including a motor and a bionic pump of the present invention to obtain extracorporeal circulation power, and then is connected to a blood circulation system composed of a microfluidic chipset.
  • CTC cells were stained with CK8 and CD45 and observed under a fluorescence microscope. CK8 (+) and CD45 (-) indicate that the cells are tumor cells, demonstrating device capture efficiency.
  • the invention can be used for online large-capacity detection of circulating tumor cells in the peripheral blood of breast cancer patients.
  • the detection sensitivity of circulating tumor cells of breast cancer can be improved, especially by using a microfluidic chipset, which can significantly improve Detection sensitivity of breast cancer circulating tumor cells.
  • the specific cytokeratin CK, leukocyte common antigen CD45, cell nucleus and cell morphology staining of circulating tumor cells were immunologically identified to effectively detect the peripheral blood of breast cancer patients. Epithelial-derived circulating tumor cells.

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Abstract

本发明公开了稀有细胞捕获系统及其应用，该系统包括流体管道设备，循环动力装置设备，组分捕获设备和任选的抗凝剂释放设备；其中该循环动力装置设备和组分捕获设备通过流体管道设备串联接入流体循环系统，形成体外流体循环通路；其中该组分捕获设备包括微流控芯片或芯片组。本发明还公开了使用所述捕获系统捕获血液中稀有细胞的方法。该系统及其应用具有大容量、在线式、低溶血性等优点。

Description

稀有细胞捕获系统及其应用 技术领域
本发明涉及生物与病理体液检测，尤其涉及一种从生物体液样本中分离稀有组分的系统及其应用，进一步涉及一种从全血细胞群体中分离稀有细胞的系统及其应用，特别涉及一种大容量、在线式、低溶血性从血液中收集循环肿瘤细胞的系统及其应用。
背景技术
稀有细胞是指生物体液样本(包括血液、胸水、腹水、尿液、脑脊液等)中的一些非典型细胞。研究表明，对稀有细胞的收集及利用其完成NGS分析，对找到疾病潜在治疗机理、病理机制及靶向药物开发具有重要指导意义。目前血液中稀有细胞检测研究方法主要有流式分选技术、形态分离方法、密度梯度离心法、膜过滤法和免疫磁性分离技术，如BD FACSAris可以实现细胞高速分选，但流式分选的瞬时激光将损伤所分选细胞，分选活性后细胞活性受损。循环肿瘤细胞(circulating tumor cell,CTCs)是指进入人体外周血的肿瘤细胞。虽然肿瘤患者外周血中CTCs数目极少，一般约10 7数量级血细胞中的CTCs仅为个位数(2-10个/ml)，但是CTCs是一种极重要的液体活检、一种判断预后及治疗间期随访的工具。由于循环肿瘤细胞数量极其稀少，对其检测的精准性和特异性要求颇高，而对其进一步的分析就更加困难，因此急需发展能够高效、大通量、快速的循环肿瘤细胞从血液样本中分离出来的便携方法与工具。
人外周血中的循环肿瘤细胞是肿瘤病灶播散进入外周血循环的肿瘤细胞(CTCs)或细胞团(CTM)，幸存的CTCs或CTM离开血液循环进入到继发脏器的局部微环境，在各类生长因子的作用下增殖生长并最终可形成转移灶。循环肿瘤细胞是肿瘤血道转移灶的重要来源，而远处转移是导致肿瘤治疗失败、复发及死亡的直接原因之一，然而，只要早期发现和干预将可能大幅降低复发及转移率，因此从血液中检测循环肿瘤细胞越来越引起人们的重视；通过对CTCs的捕获及分析，可以辅助临床医生进行肿瘤转移复发预测以及早期预警，进行患者总生存期(OS)和无进展生存期(PFS)的评估、临床放化疗的疗效监测等，进而具有指导个体化医疗、改善肿瘤患者的生存状态的重要临床意义。
如何更高效和准确的运用循环肿瘤细胞负荷作为潜在预测指标来指导实体瘤患者的病程分期和复发监控，甚至通过CTCs培养获得对化疗药物敏感性信息，已成为癌症研究中一个热门及重要的课题。
但是，要想实现临床诊断或者对CTC进行实验室分析的前提是能够获取足够的CTC细胞。由于CTCs在外周血中每10 6～10 7个单核细胞仅含有1个CTCs，因此对CTCs检测技术的灵敏度和特异度均提出了极高要求。目前各种CTCs的检测方案主要包括CTCs分离和富集系统，以及CTCs检测和鉴定系统。
其中CellSearch TM系统是最具代表性的CTCs分离收集系统，已被美国FDA批准应用于检测转移性乳腺癌循环肿瘤细胞的检测，CellSearch TM系统运用标记有针对EpCAM抗体的磁性颗粒进行血液中循环肿瘤细胞的捕获，但该系统仅对抽取的10ml血液进行检测，相对于全身循环血液，更多潜在的循环肿瘤细胞没有被检测，存在样本的选择性偏移，且该方 法检测的样本已在体外静置数小时，细胞处于低氧甚至缺氧状态，此时捕获的循环肿瘤细胞已丧失活性，难以进行信号通路与功能性等分析，无法对其进行体外培养、单细胞测序等工作。
近年来通过将微流控芯片技术与抗体捕获相结合发展出了一系列的循环肿瘤细胞芯片捕获技术。微流控细胞免疫芯片的方法是通过微流控装置(micro-fluid device)检测CTCs，该检测方法具有极高的特异性、敏感性、可重复性等特点，捕获的CTCs具有细胞活性，可分离，并用于细胞培养和其他各种下游技术研究，是CTCs临床应用价值研究的一种全新高效的方法。但是普通微流控细胞免疫芯片每次检测的血样体积较小，仍然存在样本的选择性偏移等缺陷。
CTC的识别和表征可为研究、监测和最终改变转移过程提供了机会。然而，从血液中分离CTC而不造成破裂或其他损伤是困难的。大多数现有微流体系统的主要限制是，装置效率受到装置施加在血细胞和CTC上的剪切应力的限制。太大的剪切应力将破坏血细胞和CTC。当CTC破裂时，目标分离物(CTC本身)流失，CTC内部组分散落到流体中。当血细胞(主要是白细胞)破裂释放的细胞内物质进一步复杂化血液内的物种间的非特异性相互作用,使高纯度CTC提取变得更加困难。一些现有装置已经试图通过使用低通道微流体装置来减小剪切应力来避免破坏细胞，包括血细胞和CTC。然而，低通道导致流过通道的流体体积减小，进而导致非常低的流速。这样的装置不足以快速处理大量的血液以使其实用和有用。鉴于血液中未损伤CTC的极低丰度，快速处理大量(7-30mL)的血液是高产率分离完整CTC的先决条件。目前无论哪种体外CTCs收集分离装置，均不能对全身血液进行全筛选分析，均存在血液采集样本量少，CTCs收集数量不足，样本可能存在选择性偏移等问题。将现有的外置的CTCs收集分离装置在线接入体内血液循环则可能导致对血液中细胞的损伤，存在溶血的风险，装置造成溶血后的血液会产生细胞碎片促进凝血及血液质量下降，从而可能导致潜在的临床风险。因此，需要一种能够接入体内血液循环，在线收集CTCs，同时降低溶血风险，使得处理后的血液符合血液回输标准(1、无严重溶血；2、体外时间不超过6小时；3、不能使用能使红细胞溶血的液体清洗或处理血液)的，简单易行的，在线式大容量、低溶血循环肿瘤细胞收集系统。
本发明的发明人经过创造性劳动，提供了一种满足上述标准的肿瘤细胞收集系统。上述系统具有下列优点：1、在保障CTC捕获效率的同时，保障血液流动力学及血液中细胞状态，满足血液回输标准；2、采用对微流控芯片小范围给与抗凝剂，与全身抗凝相比减少抗凝剂总使用量，降低全身抗凝的风险；3、可以在线接入血液循环系统，扩大血液样本分析量，甚至可以对全身血液进行CTCs细胞扫描，大大降低样本选择性偏移造成的系统误差；4、采用改进的微流控芯片技术，降低对血细胞的损伤，降低溶血风险；5、通过模拟心脏收缩的仿生泵为系统提供循环动力，减少对血细胞的挤压和剪切；6、可以将通过基于微流控芯片技术捕获到的循环肿瘤细胞无损的取出，进行分析或体外培养，将能更体现循环肿瘤细胞在肿瘤复发、耐药预测等发面的作用；7、本发明的系统不仅可以在线体外循环捕获稀有细胞，也可以离线实现流体的体外循环，从而提高捕获效率。8、本发明的系统不仅可以用于捕获血液中的稀有 细胞，还可以用于捕获其他生物流体中的各种细胞，或者细胞组分。
发明内容
本发明的目的是提供一种在线式大容量循环稀有细胞捕获系统及其应用。
在一方面，本发明提供了一种流体组分捕获系统，所述系统包括流体管道设备，循环动力装置设备，组分捕获设备和任选的抗凝剂释放设备；其中所述循环动力装置设备和组分捕获设备通过流体管道设备串联接入流体循环系统，形成体外流体循环通路；其中所述组分捕获设备包括微流控芯片或芯片组，所述微流控芯片内部包括扰流柱阵列。
在一方面，本发明提供了一种流体组分捕获系统，所述系统包括流体管道设备，循环动力装置设备，组分捕获设备和任选的抗凝剂释放设备；其中所述循环动力装置设备和组分捕获设备通过流体管道设备串联接入流体循环系统，形成体外流体循环通路；其中所述组分捕获设备包括微流控芯片或芯片组，所述微流控芯片内部转角均为平滑过渡的曲面。
在一方面，本发明提供了一种流体组分捕获系统，所述系统包括流体管道设备，循环动力装置设备，组分捕获设备和任选的抗凝剂释放设备；其中所述循环动力装置设备和组分捕获设备通过流体管道设备串联接入流体循环系统，形成体外流体循环通路；其中所述组分捕获设备包括微流控芯片或芯片组；其中所述循环动力装置设备包括模拟心脏挤压的仿生泵。
在一方面，本发明提供了一种微流控芯片。
在一方面，本发明提高了一种循环动力装置，所述装置包括模仿心脏挤压的仿生泵，为体外流体循环提供动力。
根据前述方面，所述组分是循环稀有细胞，优选为循环肿瘤细胞。
根据前述任一方面，其中所述流体管道设备还包括与组分捕获设备并联的旁路管道，任选地，所述旁路管道上设置有可以调节流速的限流阀或限速阀，优选所述限流阀为三通阀。
根据前述任一方面，其中在循环动力装置设备和组分捕获设备之间设置有抗凝剂释放设备，所述抗凝剂释放设备包括抗凝剂缓控释放器，任选地，所述抗凝剂缓控释放器通过三通阀接入流体管道。
根据前述任一方面，其中所述微流控芯片主体包括由下而上依次设置的基片层和盖片层，所述基片层和盖片层之间设有组分捕获室，所述盖片层上设有与所述组分捕获室连通的流体入口和流体出口，所述组分捕获室分为缓冲区和泳道部分，所述泳道中沿流动方向设置有扰流柱阵列，其中所述组分捕获室内的转角表面均为平滑过渡的曲面，本领域技术人员可以理解，所述转角既包括内凹的转角，例如所述组分捕获室的上下壁与侧壁之间的连接处，分流块或扰流柱与上下壁的连接处等，也包括外凸的转角，例如分流块或扰流柱在流体流动方向上外凸的角度。根据前述任一方面，其中所述缓冲区在靠近入口和出口处，为泳道汇合处，具有降低流速及缓冲作用。
根据前述任一方面，其中所述泳道通过分流块分为两个或者两个以上平行设置的泳道，优选地，每个泳道中包含5-500个扰流柱，优选10-200，进一步优选15-100个，例如15、16、17、18、19、20、21、22、23、24、25个，最优选为17个；任选地，所述扰流柱在泳道中按照特定间距平行排列，所述间距为0.1-5个，优选0.5-1个扰流柱的直径(长径或短 径)。
根据前述任一方面，其中所述分流块两端分别构成泳道的进出口的部分，所述进出口表面转角经防溶血处理为平滑过渡的曲面，任选地，进出口的截面可以是圆弧形、倒圆形、圆角矩形、圆角梯形等。
根据前述任一方面，所述扰流柱在流动方向上的转角表面为平滑过渡的曲面。
根据前述任一方面，其中在所述组分捕获室的内表面和扰流柱表面负载有与流体中的目标组分结合的捕获配体，优选所述捕获配体是链霉亲和素-生物素-EPCAM复合物。
根据前述任一方面，所述基片或者盖片的材料选自硅、玻璃、硅化玻璃、PDMS，或是选自聚丙烯、环烯烃共聚物、环烯烃聚合物、聚甲基丙烯酸甲酯和聚碳酸酯中的一种或两种以上的高分子聚合物材料。
根据前述任一方面，其中所述循环动力装置设备包括模仿心脏挤压的仿生泵，为体外流体循环提供动力，所述仿生泵包括弹性内胆，其两端设有入口和出口；扭转件，其套设在所述弹性内胆的外周；驱动组件，其包括动力输出构件，所述动力输出构件与所述扭转件固定连接，以便使所述动力输出构件旋转时能带动所述扭转件扭转，沿弹性内胆径向挤压或释放所述弹性内胆，优选地，在弹性内胆的入口和出口处连接单向阀，模拟心脏瓣膜的功能，实现血液的单向流动。
根据前述任一方面，其中所述扭转件包括螺旋绕制的扭转件主体，所述扭转件主体的第一端固定设置，所述扭转件主体的第二端与所述动力输出构件固定连接；或者所述扭转件包括扭转件主体，所述扭转件主体包括多条平行围设以形成柱状结构的杆件，所述扭转件主体的第一端固定设置，所述扭转件主体的第二端与所述动力输出构件固定连接。
根据前述任一方面，其中所述扭转件还包括蒙附在所述扭转件主体内周的第一保护层；并且/或者所述扭转件还包括蒙附在所述扭转件主体外周的第二保护层。
根据前述任一方面，其中所述循环动力装置设备中所述动力输出构件包括电机、调速器和换向机构，优选所述调速器是全自动正反转直流电机调速器，优选所述换向机构是锥齿轮组。
根据前述任一方面，其中通过全自动正反转直流电机调速器控制电机正反转间隔时间及转速，所述设定参数包括：所述电机正反转的间隔时间为0.5～1秒，电机转速为30-60次/分。
在一方面，本发明涉及一种血液细胞采集系统的动力装置，其特征在于，所述动力装置包括：
弹性内胆，其两端设有入口和出口；
扭转件，其套设在所述弹性内胆的外周；
驱动组件，其包括动力输出构件，所述动力输出构件与所述扭转件固定连接，以便使所述动力输出构件旋转时能带动所述扭转件扭转以挤压或释放所述弹性内胆。
根据前述任一方面，其特征在于，所述扭转件包括螺旋绕制的扭转件主体，所述扭转件主体的第一端固定设置，所述扭转件主体的第二端与所述动力输出构件固定连接；或者
所述扭转件包括扭转件主体，所述扭转件主体包括多条平行围设以形成柱状结构的杆件，所述扭转件主体的第一端固定设置，所述扭转件主体的第二端与所述动力输出构件固定连接。
根据前述任一方面，其特征在于，所述扭转件还包括蒙附在所述扭转件主体内周的第一保护层；并且/或者所述扭转件还包括蒙附在所述扭转件主体外周的第二保护层。
根据前述任一方面，其特征在于，所述动力输出构件包括与动力输出轴线垂直的端面，所述扭转件主体的第二端与所述端面固定连接。
根据前述任一方面，其特征在于，所述动力输出构件包括电机和换向机构，所述换向机构连接到所述电机，用于改变所述电机的动力输出方向。
根据前述任一方面，其特征在于，所述换向机构是锥齿轮组，所述电机与所述锥齿轮组的主动轮连接，所述端面位于所述锥齿轮组的从动轮上。
根据前述任一方面，其特征在于，所述动力输出构件包括电机和与所述电机的输出轴连接的法兰盘，所述端面位于所述法兰盘上。
根据前述任一方面，其特征在于，所述驱动组件还包括控制器，所述控制器与所述电机电连接以控制所述电机执行设定参数下的正反转操作。
一种捕获血液中稀有细胞的方法，包括使用前述任一方面的流体组分捕获系统捕获血液中稀有细胞。
根据前述方面的方法，其包括下列步骤：
(1)使前述任一方面的流体组分捕获系统的流体管道一端与采血装置连接；
(2)使微流控芯片流出的流体通过流体管道与输血装置连接；
(3)通过与微流控芯片串联或者并联设置的限流阀调节流速；
(4)使采血装置流出的血液在流体管道中经过循环动力装置，然后进入微流控芯片进行细胞捕获。
根据前述方面的方法，所述采血装置可以是采血针或者血液容器，所述输血装置可以是输血针或者与采血装置相同或不同的血液容器。
附图说明
图1流体组分捕获系统示意图，其中11是弹性内胆，111是动力装置入口，112是动力装置出口，2是流体管道，3是细胞捕获设备，4是人体，5是抗凝剂释放设备。
图2循环动力装置设备示意图，图2A是扭转件的一种实施方案；图2B是循环动力装置设备一种实施方案的示意图，其中1是循环动力装置，2是流体管道，11是弹性内胆，12是扭转件主体，13是法兰盘，14是电机，15是单向阀，16是第二保护层，112是动力装置出口；图2C是循环动力装置设备另一种实施方案的示意图，其中12是扭转件主体，13是法兰盘。
图3微流控芯片及其连接示意图，图3A微流控芯片一种实施方案的示意图，图3B是微流控芯片组及旁路管道连接示意图。
图4分光光度法检测溶血情况。
图5循环肿瘤细胞采集图。
具体实施方式
如本文所用，“稀有细胞”稀有细胞是指生物体液样本(包括血液、胸水、腹水、尿液、脑脊液等)中的一些非典型细胞。稀有细胞的例子包括但不限于循环肿瘤细胞(CTC)、循环内皮细胞(CEC)、循环多发性骨髓瘤细胞(CMMC)和循环黑素瘤细胞(CMC)。优选的稀有细胞为CTC和CEC，特别优选的稀有细胞为CTC。“循环肿瘤细胞”(CTC)是指在受试者的循环血液中检测到的癌细胞。
“分析物”是指作为检测、分离、浓缩或提取的方法的目标的流体中的分子或组分。示例性分析物包括细胞、病毒、核酸、蛋白质、碳水化合物和有机小分子。
“血液组分”是指全血的任何组分，包括宿主红细胞、白细胞和血小板。血液组分还包括血浆组分，例如蛋白质、脂质、核酸和碳水化合物，以及例如由于当前或过去怀孕、器官移植或感染而可能存在于血液中的任何其他细胞，包括稀有细胞。
“流体”或“生物流体”意在包括天然流体(例如血液、淋巴、脑脊液、尿、子宫颈灌洗液、唾液和水样品)，这些流体的一部分和已经引入细胞的流体(例如，培养基和液态组织样品)。该术语还包括裂解物。
“捕获单元”或“捕获配体”根据情况可能是指用于结合分析物的化学样品、或者全细胞凭借的其表面的组分结合物质。捕获单元可以是偶联到表面的化合物或构成表面的材料。典型的捕获单元包括抗体、寡核苷酸或多肽、核酸、其他蛋白质、合成聚合物和碳水化合物。
“通道”或“泳道”是指流体可以流过的间隙。通道可以是疏水表面上的毛细管、导管或含水液体可被限制的疏水表面的亲水纹理。
细胞的“组分”是指能够在细胞溶解液中分离到的任何组分。细胞组分可以是细胞器(例如，细胞核、近核区室、核膜、线粒体、叶绿体或细胞膜)、聚合物或分子复合物(例如，脂质、多糖、蛋白质(膜、跨膜或细胞质)、核酸(天然的、治疗性的或病原性的)、病毒颗粒或核糖体)或其他分子(例如，激素、离子、辅因子或药物)。细胞样品的“组分”是指样品中包含的细胞组分子集。
“富集的样品”是指含有这样分析物的样品：相对于通常存在的样品，其已经被处理从而增加分析物的相对含量。例如，可以通过将目标分析物的量增加至少10％、25％、50％、75％、100％或至少10、100、1000、10,000、100,000或1,000,000倍的系数来富集样品。
“剖面”是指轮廓侧视图图像。
“稀有量”的细胞指少于100个细胞/毫升流体、少于10个细胞/毫升流体、或甚至少于1个细胞/毫升流体。
其他特征和优点将从以下描述和权利要求显而易见。
为了实现发明目的，本发明人经过创造性劳动获得了流体细胞捕获系统的技术方案，所述系统包括流体管道设备，细胞捕获设备，循环动力装置设备；其中流体管道设备可以接入流体循环系统，形成体外流体循环通路，细胞捕获设备与流体管道串联或并联连接，使得流体可以从细胞捕获设备经过，从而捕获流体中含有的细胞；循环动力装置设备为体外流体循环提供动力。任选地，所述流体细胞捕获系统还包括抗凝剂释放设备，所述抗凝剂释放设备 是抗凝剂缓控释放器，所述释放器具备流量控制功能的输液泵，抗凝剂包括但不限于EDTA、柠檬酸或肝素等常见抗凝剂。优选地，所述流体是血液。优选地，所述细胞是循环稀有细胞，进一步优选地，所述细胞是循环肿瘤细胞。所述细胞捕获设备包括微流控芯片或芯片组。
在一个实施方案中，所述芯片主体包括由下而上依次设置的基片层和盖片层，所述基片层和盖片层之间设有组分捕获室，所述盖片层上设有与所述组分捕获室连通的流体入口和流体出口，所述组分捕获室分为缓冲区和泳道部分，缓冲区在靠近入口和出口处，为泳道汇合处，具有降低流速及缓冲作用，泳道部分包括一个或多个泳道，优选通过分流块分为至少两个泳道，泳道中沿流动方向设置有扰流柱阵列。
在一个实施方案中，所述分流块两端分别构成泳道的进出口的部分，所述进出口转角表面经防溶血处理为平滑过渡的曲面，例如，进出口的截面可以是圆弧形、倒圆形、圆角矩形、圆角梯形等。
在一个实施方案中，所述扰流柱横截面为流线型、纺锤形、哑铃形或者类似两个水滴尾尾相接形状使得其在流动方向上的表面为平滑过渡的曲面，从而减少机械剪切力对流体内细胞的损伤。扰流柱阵列高度等于芯片组分捕获室的内部高度。
在一个实施方案中，扰流柱设置于盖片靠近基片的一侧。
在一个实施方案中，扰流柱设置于基片靠近盖片的一侧。
在一个实施方案中，每个泳道直径3mm-5mm，高度50-100μm，扰流柱间距0.1-5个，优选0.5-1个扰流柱的直径，扰流柱阵列高度为10-100μm，每个泳道中包含50-500个扰流柱，形成微阵列。
在一个实施方案中，所述泳道平行设置。
在一个实施方案中，所述基片的长度为5-100mm，优选20-80mm，进一步优选30-60mm，最优选50mm；宽度为5-50mm，优选10-30mm，最优选20mm。
在一个实施方案中，所述泳道的数量为1-20条，优选4-15条，进一步优选6-10条，最优选8条。
在一个实施方案中，所述泳道的长度为5-100mm，优选20-40mm，最优选30mm；宽度为0.1-50mm，优选0.5-5mm，进一步优选1-3mm，最优选1.5mm；高度为0.05-0.5mm，优选0.05-0.1mm。
在一个实施方案中，所述基片或者盖片的材料为硅、玻璃、硅化玻璃、PDMS等，还可以是选自聚丙烯、环烯烃共聚物、环烯烃聚合物、聚甲基丙烯酸甲酯和聚碳酸酯中的一种或两种以上的高分子聚合物材料。
在一个实施方案中，细胞捕获设备包括芯片组合器，所述芯片组合器是医用级连接管道和卡槽，用于安放微流控芯片，使多个芯片并联或串联连接成芯片组。
在一个实施方案中，在所述组分捕获室的内表面和扰流柱表面负载链霉亲和素(Streptavidin)，该链霉亲和素能够与上皮细胞粘附分子(EPCAM)-生物素(Biotin)复合物中的标记生物素特异性结合，该EPCAM-生物素复合物能够与循环肿瘤细胞表面独特抗原特异性结合，其中链霉亲和素-生物素-EPCAM复合物中链霉亲和素-生物素复合体的链接 存在被高浓度生物素竞争洗脱的能力。任选地，也可以在组分捕获室的内表面和扰流柱表面还可以包被其他捕获配体，例如抗原、抗体、蛋白A、蛋白G、凝集素等。
在一个实施方案中，所述循环动力装置设备包括弹性内胆，其两端设有入口和出口；扭转件，其套设在所述弹性内胆的外周；驱动组件，其包括动力输出构件，所述动力输出构件与所述扭转件固定连接，以便使所述动力输出构件旋转时能带动所述扭转件扭转以挤压或释放所述弹性内胆。
在一个实施方案中，所述扭转件包括螺旋绕制的扭转件主体，所述扭转件主体的第一端固定设置，所述扭转件主体的第二端与所述动力输出构件固定连接；或者所述扭转件包括扭转件主体，所述扭转件主体包括多条平行围设以形成柱状结构的杆件，所述扭转件主体的第一端固定设置，所述扭转件主体的第二端与所述动力输出构件固定连接。
在一个实施方案中，所述扭转件还包括蒙附在所述扭转件主体内周的第一保护层；并且/或者所述扭转件还包括蒙附在所述扭转件主体外周的第二保护层。
在一个实施方案中，所述动力输出构件包括与动力输出轴线垂直的端面，所述扭转件主体的第二端与所述端面固定连接。
在一个实施方案中，所述动力输出构件包括电机和全自动正反转直流电机调速器、换向机构，所述换向机构连接到所述电机，用于改变所述电机的动力输出方向。
在一个实施方案中，所述换向机构是锥齿轮组，所述电机与所述锥齿轮组的主动轮连接，所述端面位于所述锥齿轮组的从动轮上。
在一个实施方案中，所述动力输出构件包括电机和与所述电机的输出轴连接的法兰盘，所述端面位于所述法兰盘上。
在一个实施方案中，所述驱动组件还包括控制器，所述控制器与所述电机电连接以控制所述电机执行设定参数下的正反转操作。
在一个实施方案中，通过全自动正反转直流电机调速器控制电机正反转间隔时间及转速，所述设定参数包括：所述电机正反转的间隔时间为0.5～1秒，电机转速为30-60次/分。
在一个实施方案中，在扭转件主体的内周设置保护层，能有效增大扭转件主体扭转过程中与弹性内胆的接触面积，进而使弹性内胆受到的力更均匀，实现弹性内胆的均匀压缩或舒张。
在一个实施方案中，将扭转件固定在法兰盘或锥齿轮的端面上，然后将电机输出轴与法兰盘或锥齿轮连接，使得动力输出轴的轴线能与扭转件的中心线吻合，避免出现因扭转件与动力输出轴的连接点与输出轴的轴心偏置而造成振动的现象，从而使系统的动力装置实现稳定驱动。
在一个实施方案中，在弹性内胆的入口和出口处连接单向阀，模拟心脏瓣膜的功能，实现了血液的单向流动，避免在循环过程中出现血液反流现象，以更好地保护血液内的血液细胞。
本发明的动力装置，扭转件与弹性内胆为可拆卸式设计，实现了装置的模块化、小型化，与采用蠕动泵相比，作为血室的弹性内胆为循环系统中放大的、具有弹性的装置，能有效减 小血液循环过程中血流动力学剪切力的改变，避免蠕动泵对软管中的红细胞造成挤压，极大地降低了溶血风险。通过设置扭转件，利用扭转件扭转发生形变来实现对弹性内胆的挤压和释放，并通过运转在设定参数下的电机的驱动，即通过全自动正反转直流电机调速器控制电机正反转的间隔时间及转速，实现了弹性内胆以与心脏跳动相同或相近的运动模式及节奏收缩或舒张，在保障了循环肿瘤细胞捕获效率的同时，实现了动力装置的仿生驱动，使得该动力装置可模拟心脏泵血模式，避免了现有技术中因过分挤压软管造成的破坏细胞的现象，从而保证了细胞在输送过程中的完整性，降低血液收集系统在采血过程中对血液细胞的破坏，极大地降低了溶血风险。
在一个实施方案中，所述抗凝剂释放设备是抗凝剂缓控释放器，优选为微型化前端抗凝剂缓控释放器，所述前端的意思是在血液进入微流控芯片前安置微型抗凝剂缓控释放器，保证微流控芯片内的抗凝状态。
在一个实施方案中，所述缓控释放器可以采用现有技术中常规医用镇痛泵。整体装置采用医用级塑料，体现便携式及具备匀速释放肝素钠性能，保障微流控芯片组环境内抗凝。
本公开所述的微流控芯片组根据实际采血量需要，采用串联或并联方式接入本系统。血液经血管引导出后进入本公开循环动力系统获得体外循环动力，并在远端分叉为一个或多个并联导管，分别接入微流控芯片组及旁路中，并在进入微流控芯片组后设置限流阀。连接方式见图3。优选地，所述限流阀为三通限流阀。通过设置旁路管道，使得可以方便调节通过组分捕获室的流速和压力，不容易对血细胞产生较高的剪切力，防止溶血，并且不容易造成管路堵塞。
将微流控芯片或芯片组接入血液循环系统，抗凝剂缓控释放器以三通管形式接入。抗凝剂缓控释放器内是一个具有弹性的自动收缩泵，恒定速度收缩挤压出抗凝剂，保障抗凝剂匀速泵出；并在抗凝器装置内安置单向阀，阻止微流控内血液循环回流入抗凝器内；抗凝器内含肝素钠抗凝剂。设置抗凝剂缓控释放器使得抗凝剂泵出速率为定值，并根据所需采集血液总体积调整输出量；打开输注泵开关后，泵体工作，持续恒定量泵出抗凝剂。抗凝剂与循环血液混合后通过微流控芯片微通道，与表面负载的抗体接触，并利用微通道表面负载的抗体与循环肿瘤细胞表面特异性抗原的结合将循环肿瘤细胞从血液样品中捕获并固定在微通道表面上，持续循环收集1-10小时。
在一个实施方案中，通过将链霉亲和素–生物素复合体切断实现抗体以及被其捕获的循环肿瘤细胞从微通道表面释放，从而获得高纯度的循环肿瘤细胞，并在循环收集结束后用细胞清洗液清洗所述微流控芯片，从而实现对所述被捕获的循环肿瘤细胞的收集。所述细胞清洗液是含有高浓度生物素蛋白的缓冲液，根据保存的需要可以加入防腐剂。采用CD45、CK8/18荧光抗体对洗脱的CTC细胞染色，通过荧光显微镜观察，将CD45(-)、CK8/18(+)细胞群判定为CTC细胞。
本公开所述的低溶血性经过“分光光度法”检测的经抗凝后血液通过本公开芯片后的所测定的红细胞溶血情况进行验证(图4)。
与现有技术相比，本发明具有以下的优点和特点：
1、现有系统仅分析少量血液样本，相对于全身4000ml总血量，系统抽样量太小，可能造成选择性偏移错误和假阴性率，本发明抽样样本可以覆盖全身血液，解决了目前CTC收集方案无法对全身血液进行扫描检测的问题，大大降低样本选择性偏移造成的系统误差；2、相比于基于靶向多肽循环肿瘤细胞捕获方法及微流控芯片，本发明无需在收集CTC之前进行红细胞裂解，相反更是要在收集CTC过程中对红细胞进行保护；3、本发明中的链霉亲和素-生物素复合物组合可被细胞清洗液打断，能将通过基于微流控芯片技术捕获到的循环肿瘤细胞无损的取出进行进一步分析或体外培养，将能更好的发挥循环肿瘤细胞的作用。4、微流控芯片组泳道出入口及连接管道出入口均设计为光滑过渡的曲线形状，避免了系统内部结构对血液细胞造成的潜在损伤；5、扰流柱微阵列既增加了与血液接触的表面积又防止对血细胞的机械损伤，相比现有CTC检测或血液细胞清除装置，本专利设计的扰流柱芯片更适用于在线式的血液收集系统，以芯片组形式进行血液采集保障CTC高效采集，并减少了血液溶血现象发生，保障了循环后血液的质量。6本发明的系统不仅可以在线体外循环捕获稀有细胞，也可以离线实现流体的体外循环，从而提高捕获效率。7、本发明的系统不仅可以用于捕获血液中的稀有细胞，还可以用于捕获其他生物流体中的各种细胞，或者细胞组分。
实施例
下面通过实施例对本发明作进一步说明。
实施例一：乳腺癌循环肿瘤细胞捕获能力检测
1、链霉亲和素底面包被
1.1用200μL无菌PBS清洗芯片3次，确定芯片通道中没有气泡残留。
用注射器将100ul链霉亲和素蛋白注入到芯片中，然后将芯片置于湿盒中，37摄氏度包被2小时。
2、捕获抗体的包被
2.1将人源-EpCAM捕获抗体(生物素标记)添加到1×PBS无菌溶液中，混匀，制备成5μg/ml浓度抗体捕获工作液。
2.2用注射器将100μL抗体捕获工作液注入到芯片中，然后将芯片置于湿盒中，4℃孵育过夜。在整个孵育过程中，确保所有芯片流道内始终覆盖着抗体捕获工作液。
2.3用200μL的1×PBS无菌溶液清洗包被了捕获抗体的芯片3次，除去残留在芯片中的捕获抗体工作液。
2.4用200ul 5％BSA溶液27℃封闭芯片2小时。
2.5至此，芯片上捕获抗体的包被完成，并且随时可以使用。
3.循环肿瘤细胞的捕获
3.1将装置动力部分接入含10ml乳腺癌患者血液容器中。血液经血管引导出后进入本发明动力装置系统(含电机及仿生泵)获得体外循环动力，然后接入由微流控芯片组组成的血液循环系统。
3.2通过医疗级导管接入并联/串联方式组成的微流控芯片组，打开自动前端抗凝剂缓控释放器。
3.3打开动力开关，不间断循环采集细胞悬液1小时。
3.4关闭装置动力开关，关闭自动前端抗凝剂缓控释放器，循环肿瘤细胞采集结束。
3.5用200μL 1×PBS无菌溶液注入清洗芯片3次。
5.循环肿瘤细胞及溶血的观察
镜下观察收集循环肿瘤细胞数量；根据细胞形态大小及核质比判断CTC细胞(图5)。
利用“分光光度法”检测经循环后血液中红细胞溶血情况，未发现血红蛋白值异常升高(图4)。
实施例二：乳腺癌细胞抓捕效率检测
1、链霉亲和素底面包被
1.1用200μL无菌PBS清洗芯片3次，确定芯片通道中没有气泡残留。
用注射器将100ul链霉亲和素蛋白注入到芯片中，然后将芯片置于湿盒中，37摄氏度包被2小时。
2、捕获抗体的包被
2.1将人源-EpCAM捕获抗体(生物素标记)添加到1×PBS无菌溶液中，混匀，制备成5μg/ml浓度抗体捕获工作液。
2.2用注射器将100μL抗体捕获工作液注入到芯片中，然后将芯片置于湿盒中，4℃孵育过夜。在整个孵育过程中，确保所有芯片流道内始终覆盖着抗体捕获工作液。
2.3用200μL的1×PBS无菌溶液清洗包被了捕获抗体的芯片3次，除去残留在芯片中的捕获抗体工作液。
2.4用200ul 5％BSA溶液27℃封闭芯片2小时。
2.5至此，芯片上捕获抗体的包被完成，并且随时可以使用。
3.循环肿瘤细胞的捕获
3.1将装置动力部分接入MCF7乳腺癌细胞悬液系统(模拟血液系统，体积100ml，系统内细胞数量100个)。血液经血管引导出后进入本发明动力装置系统(含电机及仿生泵)获得体外循环动力，然后接入由微流控芯片组组成的血液循环系统。
3.2通过医疗级导管接入并联/串联方式组成的微流控芯片组，打开自动前端抗凝剂缓控释放器。
3.3打开动力开关，不间断循环采集细胞悬液1小时。
3.4关闭装置动力开关，关闭自动前端抗凝剂缓控释放器，循环肿瘤细胞采集结束。
4.循环肿瘤细胞的洗脱
4.1用200μL 1×PBS无菌溶液注入清洗芯片3次。
4.2镜下观察收集MCF7细胞数量；
4.2将细胞清洗液(含高浓度生物素)200μl用注射器注入芯片中，室温静置30min，使链霉亲和素-生物素复合物分离，释放出生物素-EPCAM复合物复合物。
4.3用200μL 1×无菌PBS清洗芯片3次，收集细胞清洗液。
5.循环肿瘤细胞的观察
5.1用注射器取95％乙醇200μl固定所收集到的循环肿瘤细胞30分钟。
5.2用200μL 1×无菌PBS溶液清洗3次，去除残留的检测抗体混合液。
5.3CTC细胞进行CK8和CD45荧光染色，在荧光显微镜下观察。CK8(+)和CD45(-)表示该细胞为肿瘤细胞，证明装置捕获效率。
本发明可用于在线式大容量的检测乳腺癌患者外周血液中的循环肿瘤细胞，通过尽可能多的扫描血液，提高乳腺癌的循环肿瘤细胞检测灵敏度，特别是利用微流控芯片组，显著提高了乳腺癌循环肿瘤细胞的检测灵敏度。同时，在科学研究领域和临床试验验证的基础上，利用免疫鉴定循环肿瘤细胞的特异细胞角蛋白CK、白细胞共同抗原CD45、细胞核加上细胞形态染色的方法，有效检测出乳腺癌患者外周血中上皮来源的循环肿瘤细胞。
本发明并不限于本文所示和所述的特定实施例，而是可以不脱离由说明书所限定的本发明的精神和范围的情况下作出各种变化和修改。

Claims (10)

  1. 一种流体组分捕获系统，所述系统包括流体管道设备，循环动力装置设备，组分捕获设备和任选的抗凝剂释放设备；其中所述循环动力装置设备和组分捕获设备通过流体管道设备串联接入流体循环系统，形成体外流体循环通路；其中所述组分捕获设备包括微流控芯片或芯片组，所述微流控芯片内部转角包括扰流柱阵列。
  2. 一种流体组分捕获系统，所述系统包括流体管道设备，循环动力装置设备，组分捕获设备和任选的抗凝剂释放设备；其中所述循环动力装置设备和组分捕获设备通过流体管道设备串联接入流体循环系统，形成体外流体循环通路；其中所述组分捕获设备包括微流控芯片或芯片组，所述微流控芯片内部转角均为平滑过渡的曲面。
  3. 一种流体组分捕获系统，所述系统包括流体管道设备，循环动力装置设备，组分捕获设备和任选的抗凝剂释放设备；其中所述循环动力装置设备和组分捕获设备通过流体管道设备串联接入流体循环系统，形成体外流体循环通路；其中所述组分捕获设备包括微流控芯片或芯片组；其中所述循环动力装置设备包括模拟心脏挤压的仿生泵。
  4. 根据前述权利要求任一项的系统，其中所述流体管道设备还包括与组分捕获设备并联的旁路管道，任选地，所述旁路管道上设置有可以调节流速的限流阀，优选所述限流阀为三通阀。
  5. 根据前述权利要求任一项的系统，其中在循环动力装置设备和组分捕获设备之间设置有抗凝剂释放设备，所述抗凝剂释放设备包括抗凝剂缓控释放器，任选地，所述抗凝剂缓控释放器通过三通阀接入流体管道。
  6. 根据前述权利要求任一项的系统，其中所述微流控芯片主体包括由下而上依次设置的基片层和盖片层，所述基片层和盖片层之间设有组分捕获室，所述盖片层上设有与所述组分捕获室连通的流体入口和流体出口，所述组分捕获室通过分流块分为缓冲区和泳道部分，所述泳道中沿流动方向设置有扰流柱阵列。
  7. 根据前述权利要求任一项的系统，其中在所述组分捕获室的内表面和扰流柱表面负载有与流体中的目标组分结合的捕获配体，优选所述捕获配体是链霉亲和素-生物素-抗EPCAM抗体复合物。
  8. 根据前述权利要求任一项的系统，所述仿生泵包括弹性内胆，其两端设有入口和出口；扭转件，其套设在所述弹性内胆的外周；驱动组件，其包括动力输出构件，所述动力输出构件与所述扭转件固定连接，以便使所述动力输出构件旋转时能带动所述扭转件扭转，沿弹性内胆径向挤压或释放所述弹性内胆，优选地，在弹性内胆的入口和出口处连接单向阀，模拟心脏瓣膜的功能，实现血液的单向流动。
  9. 根据前述权利要求任一项的系统，其中所述循环动力装置设备中所述动力输出构件包括电机、调速器和换向机构，优选所述调速器是全自动正反转直流电机调速器，优选所述换向机构是锥齿轮组，优选地，所述电机正反转的间隔时间为0.5～1秒，电机转速为30-60次/分。
  10. 一种捕获血液中稀有细胞的方法，包括使用前述权利要求任一项的流体组分捕获系统捕获血液中稀有细胞，任选地，所述方法包括下列步骤：
    (1)使前述权利要求任一项的流体组分捕获系统的流体管道一端与采血装置连接；
    (2)使微流控芯片流出的流体通过流体管道与输血装置连接；
    (3)通过与微流控芯片串联或者并联设置的限流阀调节流速；
    (4)使采血装置流出的血液在流体管道中经过循环动力装置，然后进入微流控芯片进行细胞捕获。
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