CN110540933A - Circulating rare cell integrated microfluidic separation device and method - Google Patents
Circulating rare cell integrated microfluidic separation device and method Download PDFInfo
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- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
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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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/06—Magnetic means
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- 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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- 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
- C12N5/0634—Cells from the blood or the immune system
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- 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
- C12N2509/10—Mechanical dissociation
Abstract
the invention discloses a circulating rare cell integrated microfluidic separation device and a method independent of antibodies, which have simple and reasonable structural design, high processing flux, high separation efficiency and high separation purity, and integrate a circulating rare cell integrated microfluidic separation device by combining the magnetic negative sorting of cell-labeled magnetic beads with the positive sorting principle of the fluid inertia force of a spiral flow channel, wherein the device comprises a sample reaction tube, a microfluidic chip connected with the sample reaction tube, a clamping carrier for bearing and fixing the microfluidic chip and a movable magnet arranged in the clamping carrier; the sample reaction tube includes: the device comprises a sample tube bracket, a micro oscillator arranged on the sample tube bracket, a sample magnetic reaction area arranged on the micro oscillator and a sample tube for connection; the micro-fluidic chip comprises a basal layer and a chip layer arranged on the basal layer, wherein the chip layer sequentially comprises a sample inlet, a single-spiral type runner primary separation area, a magnetic separation secondary separation area and an outlet.
Description
Technical Field
the invention relates to the technical field of sorting of circulating rare cells, in particular to a circulating rare cell integrated microfluidic separation device and method.
Background
circulating Rare Cells (CRCs) include three subtypes, namely Circulating Tumor Cells (CTCs), Circulating Stem Cells (CSCs), and Circulating Fetal Cells (CFCs), wherein CTCs are the most widely used subtypes of CRCs. The separation and enrichment of the CRCs are mainly carried out under the macroscopic scale, such as density gradient centrifugal separation, fluorescence or magnetic bead based antibody labeling separation and the like. The technology has the advantages of low recovery rate of the sorted CRCs, large sample consumption and low processing flux. The microfluidic technology integrates basic operation units related to the fields of biology, medicine and chemistry, such as reaction, separation, detection and the like, on a chip with a square centimeter level, forms a fluid network by a micro-channel, and controls samples with microliter and milliliter levels through a flow channel with a micron level, so that the consumption of samples is low, and CRCs can be continuously obtained with high flux.
Common CRCs microfluidic separation technologies mainly include microstructural filtration microfluidic separation, dielectrophoresis microfluidic separation, affinity microfluidic separation and the like. The pure microstructure filtration realizes the screening of CRCs by changing the microstructure aperture according to the cell size difference, but the method has low treatment flux, easy blockage and low separation efficiency. Dielectrophoresis microfluidic separation is a method for applying a non-uniform electric field around CRCs to generate dipoles to move and separate the CRCs, and the method has the disadvantages of complex operation, complex process and low processing flux, and the electrolytic effect and the joule heating effect generated by the electric field are very easy to generate adverse effects on the cell activity and the physiological characteristics thereof, thereby being not beneficial to clinical application. The affinity microfluidic separation is based on the difference of the cell biological characteristics of the CRCs, the separation of the CRCs is realized by a specific antibody immunoaffinity separation method fixed in a microfluidic device, the separation efficiency of the CRCs with specific antigen expression is improved, the CRCs can be directly captured, higher cell activity can be kept, the cell activity is still limited to the recognition of cell epitope, and the problem of missed detection of the CRCs with non-antigen expression or weak expression exists.
Chinese patent document 1(CN 106076441a, application No. 201610398852.X) discloses a microfluidic device for size-based detection of circulating tumor cells, comprising: the device comprises a solution storage chamber, a micro-fluidic chip, a waste liquid collecting needle cylinder and a power system which are connected in sequence. The device still realizes the separation of single CTCs by a multi-stage block filtering area formed by column arrays with different intervals, the blood sample processing flux is only 10 mL/h-18 mL/h, although the problem of easy blockage is solved, the defects of low separation efficiency and low detection flux still exist, and the method based on cell size can miss the detection of small CTCs.
Chinese patent document 2(CN 107084916a, application No. 201710195836.5) discloses a microfluidic chip device for separating circulating tumor cells, which includes a microfluidic chip and a chip clamp, where the microfluidic chip includes a chip inlet, a single-helix chip, a rare cell collection channel, and a leukocyte outlet. The method is based on the difference between the fluid inertia force and the nuclear-to-cytoplasmic ratio and the surface charge of circulating tumor cells and blood cells, and collects rare cells with different particle sizes through 3 outlets at the tail end of a single-spiral microfluidic channel. The essence of the method is still a method for separating by migrating and converging CTCs to a specific fluid balance position under the action of fluid inertia force (fluid inertia lift force and Dean flow drag force) of a spiral flow channel by utilizing the difference of the sizes of the CTCs and other cells and assisted by charge difference, although the processing flux is high and can reach 60-200ml/h, the defect of low purity of separated CTCs exists.
Disclosure of Invention
The invention aims to provide the antibody-independent circulating rare cell integrated microfluidic separation device and method which have the advantages of simple and reasonable structural design, high processing flux, high separation efficiency and high separation purity, and overcome the defects of small processing flux, low separation purity, antibody dependence and low integration degree of the conventional CRCs separation technology.
The technical scheme adopted by the invention for solving the problems is as follows: the integrated micro-fluidic separation device for the circulating rare cells integrates a magnetic negative sorting principle of cell-labeled magnetic beads and a positive sorting principle of fluid inertia force of a spiral flow channel, and comprises a sample reaction tube (2), a micro-fluidic chip (3) connected with the sample reaction tube (2), a clamping carrier (4) for bearing and fixing the micro-fluidic chip (3) and a movable magnet (6) arranged in the clamping carrier (4);
The sample reaction tube (2) comprises: the device comprises a sample tube bracket (9), a micro oscillator (8) arranged on the sample tube bracket (9), a sample magnetic reaction area arranged on the micro oscillator (8) and a sample tube (5) for connection;
the microfluidic chip (3) comprises a substrate layer (34) and a chip layer (33) arranged on the substrate layer (34), wherein the chip layer sequentially comprises a sample inlet (31), a single-spiral-type runner primary separation area, a magnetic separation secondary separation area and an outlet (32);
The clamping carrier (4) can bear and fix the microfluidic chip (3) and comprises a chip base (41) containing a magnet adapter (42) and an elastic buckle (47) and a carrier upper cover (43) connected with the chip base (41), the carrier upper cover (43) is provided with a soft plug jack (45) and an elastic clamping piece (46) embedded into the carrier upper cover (43), the elastic clamping piece (46) is matched with the elastic buckle (47) to close the carrier upper cover (43), and the soft plug jack (45) is respectively matched and corresponding to a sample inlet (31) and an outlet (32) of the microfluidic chip; the movable magnet (6) is clamped into a magnet adapter (42) of the chip base (41), corresponds to the magnetic separation secondary separation area of the microfluidic chip, and can magnetically adsorb cells combined with magnetic beads.
The sample magnetic reaction area comprises a sample inlet (51), a magnetic bead suspension inlet (52), a reaction cavity (21) and a sample pipeline outlet (53), wherein the sample pipeline outlet (53) is connected with a sample pipe (5), and the volume of the sample magnetic reaction area is 0.5-5 ml, and the sample magnetic reaction area is used for the immune magnetic binding reaction of a processed sample and magnetic beads containing CD45 antibodies. The sample inlet (51) and the magnetic bead suspension inlet (52) are communicated with the reaction cavity (21) through a micro-flow pump (1) connected with a sample tube (5), the sample inlet (51) and the magnetic bead suspension inlet (52) inject a biological sample and a magnetic bead suspension into the reaction cavity (21) through the micro-flow pump (1) connected with the sample tube (5), and preferably, the flow rate range of the micro-flow pump is 0.03-8.2 ml/min. Preferably, the magnetic beads are superparamagnetic, uniform-sized, uniform-surface polymer magnetic beads, and more preferably magnetic Polystyrene (PS) magnetic beads.
The sample tube (5) is a plastic tube which is soft, small in surface tension, extremely low in friction coefficient, free of adhesion, non-hydrophilic, non-toxic and good in biocompatibility, and preferably a plastic tube containing Polytetrafluoroethylene (PTFE).
the micro oscillator (8) provides mixed oscillating force for a sample magnetic reaction area, the micro oscillator (8) comprises a sample magnetic reaction area joint (81) and a sample tube bracket clamping groove (82), the sample magnetic reaction area joint (81) is used for stably connecting and fixing a reaction cavity (21), a sample pipeline outlet (53) is arranged on the sample magnetic reaction area joint (81), the sample pipeline outlet (53) is connected with the sample tube (5), the sample magnetic reaction area joint (81) corresponds to the sample pipeline outlet (53), the sample tube (5) passes through the micro oscillator (8), an electromagnetic controller (83) is arranged in the sample magnetic reaction area joint (81), the electromagnetic controller (83) comprises an electromagnet (833), an armature (831) matched with the electromagnet (833) and a spring (832) arranged between the electromagnet (833) and the armature (831), the sample tube (5) at the sample pipeline outlet (53) is clamped on the spring (832), when the electromagnet (833) is electrified, the electromagnet (833) attracts the armature (831), the spring (832) is compressed, the spring (832) can clamp the sample tube (5) at the sample pipeline outlet (53) to cut off fluid in the pipeline to prevent liquid which is not reacted in the sample magnetic reaction area from flowing out, when the micro oscillator (8) stops vibrating, the electromagnet (833) is powered off, the spring (832) of the electromagnetic controller (83) can reset and rebound, the sample tube (5) at the sample pipeline outlet (53) can be opened, a fluid passage is opened, and the reacted liquid flows into the sample inlet (31) of the microfluidic chip (3); the sample tube bracket clamping groove (82) can be clamped into the sample tube bracket (9) to fixedly connect the micro oscillator (8) with the sample tube bracket (9), the sample tube bracket clamping groove (82) comprises two metal contacts (84), and the two metal contacts can be connected with two metal electrodes (91) in the sample tube bracket (9) to form a current path for supplying power to the micro oscillator. The micro oscillator (8) can fully mix and react leukocytes in the sample magnetic reaction area with CD45 antibody magnetic beads through micro vibration, so that the leukocytes and the CD45 antibody magnetic beads can be conveniently and uniformly combined, and the cells and target CRCs cannot be damaged. The electromagnetic controller (83) is provided with two power supply connecting contacts (834) and (835), and the two power supply connecting contacts (834) and (835) are connected with the two metal contacts (84) of the sample tube bracket clamping groove (82).
The sample tube bracket (9) is of a hollow structure, a sample tube (5) can penetrate out of the bracket, two electrodes (91) are arranged outside the bracket and can be connected with a sample tube bracket clamping groove metal contact (84) of a micro oscillator (8) and a contact at a soft plug jack of a clamping carrier (4) to play a role in supporting the sample tube (5), bridging a sample magnetic reaction area and the clamping carrier (4) so as to form a current and fluid passage.
preferably, the sample magnetic reaction area, the micro oscillator (8) and the sample tube bracket (9) are 3 independent structures and are integrally formed by combining and splicing, wherein the sample magnetic reaction area is a 1-time use part, and cross contamination among different samples can be effectively prevented.
The material of a basal layer (34) of the microfluidic chip (3) is preferably a glass slide, the material of a chip layer (33) is preferably Polydimethylsiloxane (PDMS), the primary separation area of the single-spiral flow channel is composed of single-spiral micro-flow channels (10) with 4 or more complete spirals (less than 20) and at least 3 branches (less than 10) at the tail ends of the spiral flow channels, preferably, the height of each single-spiral micro-flow channel (10) is 40-110 mu m, the width of each flow channel is 150-600 mu m, and the distance between the flow channels is 450-500 mu m; the chip layer sample inlet (31) is positioned in the middle of the single-spiral flow channel (10), and the distance from the first inner spiral radius (R) of the single-spiral flow channel ranges from 2.5mm to 4.5 mm.
The magnetic separation secondary separation area of the invention is composed of 3 or more (less than 10) magnetic separation tanks (35), each magnetic separation tank (35) is connected with the end branch of the spiral micro-channel (10) of the single spiral channel primary separation area, and the capacity of the single magnetic separation tank (35) is 0.2 ml-2 ml. Preferably, the magnetic separation pool (35) is circular or elliptical, so that the CRCs can be magnetically separated to the maximum extent, and blockage can be effectively prevented. The outlet (32) is positioned at the tail end of the magnetic separation secondary separation area, 3 or more (less than 10) outlets (32) correspond to the magnetic separation pool (35), and the corresponding number of cell collection tubes (7) are arranged at the outlets (32) for collecting CRCs and other cells. High purity CRCs can be collected when the moving magnet (6) is present, and cells attached to magnetic beads can be collected when the moving magnet (6) is removed.
The magnet adapters (42) on the chip base (41) of the carrier are matched and arranged into circular grooves or elliptical grooves with corresponding quantity according to the shape and the quantity of the magnetic separation tanks (35), the depth of the circular grooves is equal to the height of the moving magnets placed in the circular grooves, and the moving magnets (6) can be fixed; the carrier upper cover (43) is connected with the chip base (41) through a movable connecting seat (44).
the inner pipeline of the soft plug (11) is of a 1-shaped through hole structure and is divided into an inlet plug and an outlet plug, the inlet plug can be tightly butted with a chip sample inlet to form a fluid passage, and liquid leakage is effectively prevented; the outlet plug is tightly butted with the chip outlet, and the outlet of the outlet plug is connected with a sample tube which can be connected into a cell collecting tube, so that corresponding cells can conveniently flow into and be collected. The soft plug can be accurately inserted into a soft plug jack (45) of the upper cover of the clamping carrier so as to be tightly connected with the chip sample inlet (31), the outlet (32) and the clamping carrier (4) to form a fluid passage. Preferably, the soft plug (11) is made of plastic with an extremely low friction coefficient, no adhesion, no hydrophilicity, no toxicity, good biocompatibility and good sealing performance, and the plastic is more preferably PTFE. In order to avoid biological cross contamination, the soft competition is used for 1 time, and each minute of sample corresponds to a pair of soft plugs.
the moving magnet (6) is of a semicircular or semielliptical ring structure, is well matched with the magnet adapter (42) on the chip base (41), and can enable white blood cells with magnetic beads to form annular or semielliptical ring-shaped colonies without gathering at the inlet and the outlet (32) of the magnetic separation pool, so that a gap is reserved, and the blockage of the inlet and the outlet caused by cell gathering is well avoided. The movable magnet (6) can be put in or taken out according to the cell collection requirement, when the movable magnet (6) is put in the adapter (42), high-purity CRCs can be collected, after the CRCs are collected, the movable magnet (6) is removed, and the white blood cells connected with magnetic beads can be recovered.
A circulating rare cell integrated microfluidic separation method adopts a circulating rare cell integrated microfluidic separation device, and comprises the following steps:
1) placing the micro-fluidic chip (3) into a clamping carrier (4);
2) Loading a sample magnetic reaction area on a sample magnetic reaction area connector (81) of a micro oscillator (8), connecting a sample tube bracket clamping groove (82) with a sample tube bracket (9), butting a metal electrode (91) on the sample tube bracket (9) with a metal contact (84) during connection, finally connecting a clamping carrier (4) and starting a power supply, starting the micro oscillator (8) to vibrate, attracting an armature (831) by an electromagnet (833) after the electromagnetic controller (83) is electrified, compressing a spring (832), clamping a sample tube (5) at a sample tube outlet (53) by the spring (832) to cut off fluid in the tube to prevent liquid which is not reacted in the sample magnetic reaction area from flowing out;
3) The method comprises the following steps that a monocyte layer (PBMCs) subjected to density gradient centrifugation treatment is added into a reaction cavity (21) of a sample magnetic reaction area through a sample inlet (51) by a micro-flow pump (1), meanwhile, a magnetic bead suspension is injected into the reaction cavity (21) through a magnetic bead suspension inlet (52), and the two are mixed and combined to react for 10-30 min (preferably 20 min);
4) The micro-oscillator (8) is closed, the electromagnet (833) of the electromagnetic controller (83) is powered off, the spring (832) of the electromagnetic controller (83) resets and rebounds, the sample tube (5) at the outlet (53) of the sample pipeline is opened, and the reaction completion liquid enters the microfluidic chip (3) through the outlet (53) of the sample pipeline to carry out cell separation;
5) The reaction finished liquid enters a single-spiral micro-flow channel (10) of a single-spiral flow channel primary separation area to be separated by fluid inertia force under the action of a micro-flow pump (1), cells with the cell diameter larger than 20 mu m separated by the fluid inertia force enter a first magnetic separation pool from an inner measuring flow channel, cells with the cell diameter of 15 +/-5 mu m and the medium particle size enter a second magnetic separation pool from a middle flow channel, cells with the cell diameter smaller than or equal to 7 mu m enter a third magnetic separation pool from an outer flow channel, under the action of a magnetic field of a moving magnet positioned below the magnetic separation pools, leukocytes connected with magnetic beads are gathered in each magnetic separation pool and separated from CRCs, the used moving magnet (6) is in a semi-ring shape, the leukocytes can also form semi-ring-shaped gathering, and the CRCs flow into a cell collection pipe (7) through an outlet (32) along a sample pipe (5) to be collected;
6) After the CRCs are collected, the microflow pump (1) is closed, the clamping carrier (4) is opened, the microflow control chip (3) is taken out, the movable magnet (6) in the chip base (41) of the clamping carrier is removed, the microflow control chip (3) which is taken out is put back into the clamping carrier (4), the microflow pump (1) is started, and the white blood cells connected with the magnetic beads are collected by a new cell collecting pipe (7).
Compared with the prior art, the invention has the following advantages and effects:
The invention effectively integrates the spiral flow channel fluid inertia force (fluid inertia lifting force and Dean flow pulling force) positive sorting chip and the cell marking magnetic bead magnetic negative sorting chip, constructs the circulating rare cell integrated microfluidic separation device, and greatly improves the circulating rare cell detection flux and purity. The invention utilizes the single-spiral micro-channel primary separation area to roughly separate out CRCs with lower purity in high flux, then greatly improves the sensitivity of white cells to a magnetic field by marking magnetic beads with white cells, further effectively removes the white cells under the action of the magnetic field of the secondary separation area, and obtains the active CRCs with high purity. The device has simple and reasonable structural design and convenient manufacture, well keeps the advantage of high processing flux of the single-spiral micro-channel chip, simultaneously further removes leucocytes through integrated magnetic negative direction separation and capture to purify CRCs, and well solves the problem of low purity of the CRCs obtained by the single-spiral micro-channel. In addition, the integrated microfluidic separation device is an antibody-independent CRCs separation system, so that the cell integrity and the cell activity of the CRCs are well maintained, and the acquired CRCs are conveniently applied to downstream protein characterization, gene analysis, cell culture and cell detection research. In conclusion, the invention provides the antibody-independent circulating rare cell integrated microfluidic separation device which has the advantages of simple and reasonable structural design, high processing flux, high separation rate and high separation purity.
Drawings
Fig. 1 is a schematic diagram of the overall structure of a circulating rare cell integrated microfluidic separation device.
FIG. 2 is a schematic view of the structure of a sample reaction tube and its partial components according to the present invention.
fig. 3 is a schematic structural diagram of a microfluidic chip according to the present invention.
Fig. 4 is a schematic structural diagram of the clamp carrier of the present invention.
FIG. 5 is a schematic view of a soft plug construction;
FIG. 6 is a schematic diagram of the structure of an electromagnetic controller according to the present invention;
In the figure: the device comprises a micro-flow pump 1, a sample reaction tube 2, a micro-fluidic chip 3, a clamping carrier 4, a sample tube 5, a moving magnet 6, a cell collecting tube 7, a micro-oscillator 8, a sample tube bracket 9, a single-spiral micro-channel 10, an inner measuring channel 10-1, a middle channel 10-2 and an outer channel 10-3; the device comprises a reaction cavity 21, a sample inlet 31, an outlet 32, a chip layer 33, a substrate layer 34, a magnetic separation pool 35, a chip base 41, a magnet adapter 42, a carrier upper cover 43, a movable connecting seat 44, a soft plug jack 45, an elastic clamping piece 46, an elastic buckle 47, a sample inlet 51, a magnetic bead suspension inlet 52, a sample pipeline outlet 53, a sample magnetic reaction area joint 81, a sample tube support clamping groove 82, an electromagnetic controller 83, a metal contact 84 and a metal electrode 91.
Detailed Description
As shown in fig. 1, 2, 3 and 4, a circulating rare cell integrated microfluidic separation device comprises a sample reaction tube 2, a microfluidic chip 3, a holding carrier 4 and a moving magnet 6, wherein the sample reaction tube 2 mainly comprises a sample magnetic reaction area, a micro oscillator 8, a sample tube 5 and a sample tube bracket 9; the micro-fluidic chip 3 is formed by combining a substrate layer 34 and a chip layer 33 arranged on the substrate layer 34, wherein the chip layer 33 comprises a sample inlet 31, a single-spiral type flow channel 10 primary separation area, a magnetic separation secondary separation area and an outlet 32; the clamping carrier 4 can bear and fix the microfluidic chip 3, the clamping carrier 4 comprises a chip base 41 containing a magnet adapter 42 and an elastic buckle 47, and a carrier upper cover 43 connected with the chip base 41, the carrier upper cover 43 is provided with a soft plug jack 45 and an elastic clamping piece 46 embedded into the carrier upper cover 43, the carrier upper cover 43 is closed, and the soft plug jack 45 is respectively matched and corresponding to the sample inlet 31 and the outlet 32 of the microfluidic chip 3; the movable magnet 6 can be accurately clamped into the magnet adapter 42 of the chip base 41, corresponds to the magnetic separation secondary separation area of the microfluidic chip 3, and can magnetically adsorb cells combined with magnetic beads.
as shown in fig. 1 and fig. 2, in the present invention, the sample magnetic reaction area includes a sample inlet 51, a magnetic bead suspension inlet 52, a reaction chamber 21 and a sample line outlet 53, the sample line outlet 53 is connected to the sample tube 5, and the sample magnetic reaction area has a volume of 0.5ml to 5ml, and is used for an immunomagnetic binding reaction between a processed sample and magnetic beads containing CD45 antibodies. The sample inlet 51 and the magnetic bead suspension inlet 52 inject the biological sample and the magnetic bead suspension into the reaction cavity 21 through the micro-flow pump 1 connected with the sample tube 5, and the flow rate of the micro-flow pump ranges from 0.03ml/min to 8.2 ml/min. The magnetic beads are superparamagnetic, uniform-sized, uniform-surface polymer magnetic beads, and more preferably magnetic Polystyrene (PS) magnetic beads.
The sample tube 5 of the present invention is a plastic tube that is soft, has a small surface tension, an extremely low friction coefficient, is non-adhesive, is not hydrophilic, is non-toxic, and has good biocompatibility, and is preferably a plastic tube containing Polytetrafluoroethylene (PTFE).
As shown in fig. 1, 2 and 6, the micro-oscillator 8 of the present invention provides a mixed oscillating force for a sample magnetic reaction area, and includes a sample magnetic reaction area joint 81 and a sample tube holder slot 82, the sample magnetic reaction area joint 81 is used for stably connecting and fixing a reaction chamber 21, a sample line outlet 53 is disposed on the sample magnetic reaction area joint 81, the sample line outlet 53 is connected with the sample tube 5, the sample magnetic reaction area joint 81 corresponds to the sample line outlet 53, so that the sample tube 5 passes through the micro-oscillator 8, an electromagnetic controller 83 is disposed in the sample magnetic reaction area joint 81, as shown in fig. 6, the electromagnetic controller 83 includes an electromagnet 833, an armature 831 cooperating with the electromagnet 833, a spring 832 disposed between the electromagnet 833 and the armature 831, and two metal contacts 834 and 835 connected with a power supply (the power supply connection contacts 834 and 835 are connected with the two metal contacts 84 in the sample tube holder slot 82), the sample tube 5 at the sample line outlet 53 is clamped on the spring 832, when the electromagnet 833 is electrified, the electromagnet 833 attracts the armature 831, the spring 832 is compressed, the spring 832 clamps the sample tube 5 at the sample line outlet 53 to cut off the fluid in the line to prevent the unreacted liquid in the sample magnetic reaction area from flowing out, and to cut off the fluid in the line to prevent the unreacted liquid in the sample magnetic reaction area from flowing out, when the micro-oscillator 8 stops vibrating, the electromagnet 833 is powered off, the spring 832 of the electromagnetic controller 83 resets and rebounds, the sample tube 5 at the sample line outlet 53 can be opened, and the fluid passage is opened to allow the reacted liquid to flow into the sample inlet of the microfluidic chip; the sample tube holder slot 82 can be clipped into the sample tube holder 9 to fixedly connect the micro-oscillator 8 and the sample tube holder 9, and the slot 82 comprises two metal contacts 84 which can be connected with two metal electrodes 91 in the sample tube holder 9 to form a current path for supplying power to the micro-oscillator. The micro-oscillator 8 can make the leucocyte in the sample magnetic reaction area and the CD45 antibody magnetic bead fully mixed and react through micro-vibration, so that the leucocyte and the CD45 antibody magnetic bead are conveniently and uniformly combined, and the cell and the target CRCs cannot be damaged.
as shown in fig. 1 and 2, the sample tube holder 9 of the present invention is a hollow structure, through which the sample tube 5 can pass, and two electrodes 91 are provided outside the holder and can be connected to the metal contacts 84 of the sample tube holder slot of the micro-oscillator 8 and the contacts at the cork holes of the clamp carrier 4, so as to support the sample tube 5 and bridge the sample magnetic reaction area and the clamp carrier 4, thereby forming current and fluid paths.
the sample magnetic reaction area, the micro oscillator 8 and the sample tube bracket 9 are 3 independent structures and are integrally formed by combination and splicing, wherein the sample magnetic reaction area is a 1-time use part, and cross contamination among different samples can be effectively prevented.
As shown in fig. 1 and fig. 3, the substrate layer 34 of the microfluidic chip 3 of the present invention is a glass slide, the chip layer 33 is Polydimethylsiloxane (PDMS), the primary separation zone of the single spiral channel is composed of single spiral microchannels 10 with 4 or more complete spirals and at least 3 branches at the ends of the spiral channels, preferably, the single spiral microchannels 10 have a height of 40 μm to 110 μm, a width of 150 μm to 600 μm, and an interval between channels of 450 μm to 500 μm; the sample inlet 31 of the chip layer 33 is positioned in the middle of the single-spiral runner 10, and the radius R from the first inner spiral of the single-spiral runner ranges from 2.5mm to 4.5 mm.
As shown in FIG. 3, the magnetic separation secondary separation zone of the present invention is composed of 3 or more magnetic separation cells 35, each magnetic separation cell 35 is connected to the terminal branch of the spiral micro flow channel 10 of the single spiral flow channel primary separation zone, and the capacity of the single magnetic separation cell 35 is 0.2 ml-2 ml. The magnetic separation tank 35 is circular or elliptical, so that the CRCs can be magnetically separated to the maximum extent, and blockage can be effectively prevented. The outlet 32 is positioned at the tail end of the magnetic separation secondary separation area, 3 or more outlets 32 are corresponding to the magnetic separation pool 35, and a corresponding number of cell collection tubes 7 are arranged at the outlets 32 for collecting CRCs and other cells. High purity CRCs can be collected when the moving magnet 6 is present, and cells attached to magnetic beads can be collected when the moving magnet 6 is removed.
As shown in fig. 4, the magnet adapters 42 on the chip base 41 of the clip carrier 4 of the present invention are configured to be circular grooves or elliptical grooves of corresponding number according to the shape and number of the magnetic separation cells 35, the depth of the circular grooves is equal to the height of the moving magnets 6 placed therein, and the moving magnets 6 can be fixed; the carrier top cover 43 is connected to the chip base 41 by a movable connecting base 44.
As shown in fig. 5, the inner pipe of the soft plug 11 of the present invention is a 1-shaped through hole structure, and is divided into an inlet plug and an outlet plug, the inlet plug can be tightly butted with the chip sample inlet 31 to form a fluid passage, so as to effectively prevent liquid leakage; the outlet plug is closely butted with the chip outlet, and the outlet of the outlet plug is connected with the sample tube 5 which can be connected into the cell collecting tube, so that the corresponding cells can conveniently flow into and be collected. The soft plug 11 can be precisely inserted into the soft plug jack 45 of the upper cover of the carrier to tightly connect and communicate the chip sample inlet 31, the outlet 32 and the carrier 4 to form a fluid passage. The soft plug 11 is made of plastic with extremely low friction coefficient, no adhesion, no hydrophilicity, no toxicity, good biocompatibility and good sealing performance, and the plastic is PTFE. In order to avoid biological cross contamination, the soft plug 11 is used for 1 time, and each sample corresponds to one pair of soft plugs.
As shown in FIG. 1 and FIG. 4, the moving magnet 6 of the present invention is a semi-circular or semi-elliptical ring structure, which is well matched with the magnet adapter 42 on the chip base 41, so that the leukocytes with magnetic beads can form a ring or semi-elliptical ring-shaped colony without gathering at the inlet and outlet 32 of the magnetic separation cell, leaving a gap, and well avoiding the inlet and outlet blockage caused by cell gathering. The movable magnet 6 can be put in or taken out according to the cell collection requirement, when the movable magnet 6 is put in the magnet adapter 42, high-purity CRCs can be collected, after the CRCs are collected, the movable magnet 6 is removed, and the white blood cells connected with the magnetic beads can be recovered.
Example 1 Cyclic rare cell Integrated microfluidic separation device to separate CRC
In the present embodiment, the present invention is further described with reference to the following specific embodiments. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
As shown in fig. 1, a microfluidic device according to a preferred embodiment of the present invention mainly includes: the microfluidic pump 1 for controlling the flow and the flow rate of the fluid is communicated with a sample reaction tube 2 through a sample tube 5, reaction completion liquid for completing the combination of cells and magnetic beads in the sample reaction tube 2 is vertically communicated with a microfluidic chip 3 through the sample tube 5 and a clamping carrier 4 loaded with a moving magnet 6 to separate CRCs, and the separated CRCs respectively flow into a cell collecting tube 7 through the sample tube 5 connected with an outlet 32 of the chip 3 to be collected. After completion of the CRC collection, the moving magnet 6 is removed from the carrier 4 and replaced with a new cell collection tube 7, and leukocytes having CD45 magnetic beads attached thereto can be recovered.
As shown in fig. 2 and 3, a circulating rare cell integrated microfluidic separation method, which adopts a circulating rare cell integrated microfluidic separation device, comprises the following steps:
1) Placing the microfluidic chip 3 into a clamping carrier 4;
2) Loading a sample magnetic reaction area on a sample magnetic reaction area joint 81 of a micro oscillator 8, connecting a sample tube bracket clamping groove 82 with a sample tube bracket 9, butting a metal electrode 91 on the sample tube bracket 9 with a metal contact 84 during connection, finally connecting a clamping carrier 4 and starting a power supply, starting the micro oscillator 8 to vibrate, attracting an armature 831 by an electromagnet 833 after an electromagnetic controller 83 is electrified, compressing a spring 832, clamping a sample tube 5 at a sample pipeline outlet 53 by the spring 832, and preventing liquid which is not reacted in the sample magnetic reaction area from flowing out by cutting off fluid in the pipeline;
3) The microfluid pump 1 leads the mononuclear cell layer (PBMCs) processed by density gradient centrifugation to pass through a sample inlet 51 and be added into a reaction cavity 21 of a sample magnetic reaction area, and simultaneously, a CD45 magnetic bead suspension (Dynabeads TM CD45, Invitrogen TM) is injected into the reaction cavity 21 through a magnetic bead suspension inlet 52, and the two are mixed and combined for reaction for 20 min;
4) The micro-oscillator 8 is closed, the electromagnet 833 of the electromagnetic controller 83 is powered off, the spring 832 of the electromagnetic controller 83 resets and rebounds, the sample tube 5 at the sample tube outlet 53 is opened, and the reaction completion liquid enters the micro-fluidic chip 3 through the sample tube outlet 53 for cell separation;
5) Under the action of the micro-flow pump 1, the reaction completion liquid enters the single-spiral micro-flow channel 10 of the single-spiral flow channel primary separation area at the flow rate of 2ml/min for fluid inertia force separation, cells with the cell diameter being more than or equal to 20 microns separated by the fluid inertia enter the magnetic separation pool 35 from the inner measuring flow channel 10-1, cells with the medium particle diameter being 15 +/-5 microns enter the magnetic separation pool 35 from the middle flow channel 10-2, and cells with the cell diameter being less than or equal to 7 microns enter the magnetic separation pool 35 from the outer side flow channel 10-3. Under the action of the magnetic field of the moving magnet positioned below the magnetic separation cells 35, the leukocytes connected with the CD45 magnetic beads are gathered in each magnetic separation cell and separated from the CRCs, and because the moving magnet 6 is in a semi-annular shape, the leukocytes also form semi-annular gathering, so that the blockage caused by gathering is effectively avoided, and the CRCs flow into the cell collection tube 7 along the sample tube 5 through the outlet 32 to be collected.
6) after the collection of the CRCs is completed, the microfluidic pump 1 is closed, the clamp carrier 4 is opened, the microfluidic chip 3 is taken out, the moving magnet 6 in the chip base 41 of the clamp carrier is removed, the taken-out microfluidic chip 3 is put back into the clamp carrier 4, the microfluidic pump 1 is started, and the leukocytes connected with CD45 magnetic beads are collected by a new cell collection tube 7.
example 2 isolation of circulating tumor cells in human peripheral blood Using the present invention
1 blood sample Collection and pretreatment
2mL of venous peripheral blood samples of breast cancer patients were collected by a blood collection tube, and the whole blood samples were diluted to 4mL with 1 XPBS at a volume ratio of 1:1, added to a centrifuge tube containing 3mL of density gradient centrifugation solution, and centrifuged at 1000 Xg for 10min at room temperature.
Removing the supernatant plasma, transferring the PBMCs layer to a new 15mL centrifuge tube, washing the PBMCs with 10mL of primary cell complete culture medium, centrifuging for 10min at 4 ℃ at 300 Xg, carefully removing the supernatant, gently resuspending the PBMCs with 1mL of primary cell complete culture medium for 2 times, transferring to a 2.5mL centrifuge tube, centrifuging for 10min at 300 Xg at room temperature, carefully removing the supernatant, sucking cell washing liquid to resuspend the PBMCs precipitate to 2mL, and separating the rare cells to be circulated by the integrated microfluidic separation device.
2 isolation of circulating tumor cells
starting a micro-flow pump 1 for controlling the flow rate and the flow rate of fluid, inserting a sample tube 5 connected with a sample inlet 51 into the pretreated PBMCs in the sample tube 1, inserting the sample tube 5 connected with a sample inlet 52 into a centrifugal tube containing 2-8 ℃ precooled CD45 magnetic bead suspension (Dynabeads CD45, Invitrogen), pumping 2ml of PBMCs suspension and 200 mul of CD45 magnetic bead suspension into a reaction cavity 21 of a sample reaction tube 2 by the micro-flow pump 1, vibrating the micro-oscillator 8 for 25min, finishing the reaction of combining white blood cells and magnetic beads, automatically closing the micro-oscillator 8, and resetting an electromagnetic controller 83.
The reaction completion liquid enters the single-spiral micro-channel 10 of the single-spiral flow channel primary separation area of the micro-fluidic chip 3 through the sample pipeline outlet 53 at the flow speed of 2ml/min under the action of the micro-fluidic pump 1 for fluid inertia force separation, cells with the cell diameter being more than or equal to 20 microns separated by the fluid inertia enter the magnetic separation pool 35 from the inner measuring flow channel 10-1, cells with the medium particle diameter being 15 +/-5 microns enter the magnetic separation pool 35 from the middle flow channel 10-2, and cells with the cell diameter being less than or equal to 7 microns enter the magnetic separation pool 35 from the outer flow channel 10-3. Under the action of the magnetic field of the moving magnet positioned below the magnetic separation cells 35, the leukocytes connected with CD45 magnetic beads are gathered in each magnetic separation cell and separated from the circulating tumor cells, and the separated circulating tumor cells with different sizes flow into the cell collection tube 7 through the outlet 32 along the sample line 5 to be collected for downstream various analyses (such as immunochemical staining, Next Generation Sequencing (NGS), Fluorescence In Situ Hybridization (FISH), and the like).
And (3) closing the micro-flow pump 1, opening the holding carrier 4, taking out the micro-flow control chip 3, removing the movable magnet 6 in the chip base 41 of the holding carrier, putting the taken-out micro-flow control chip 3 back into the holding carrier 4, starting the micro-flow pump 1, and recovering the white blood cells connected with the CD45 magnetic beads by using a new cell collecting pipe 7.
Parts not described in detail in this specification are prior art using structures and principles known to those skilled in the art.
The above description is within the reach of a person skilled in the art.
It should be noted that the specific embodiments described in this specification are only for describing the preferred embodiments of the present invention, and do not limit the scope of the present invention, and those skilled in the art should include the protection scope defined by the claims of the present patent application only by making equivalent or simple changes to the structure, features and principles described in the concept of the present invention without departing from the design of the present invention.
Claims (10)
1. The circulating rare cell integrated microfluidic separation device is characterized by comprising a sample reaction tube (2), a microfluidic chip (3) connected with the sample reaction tube (2), a clamping carrier (4) for bearing and fixing the microfluidic chip (3) and a movable magnet (6) arranged in the clamping carrier (4);
The sample reaction tube (2) comprises: the device comprises a sample tube bracket (9), a micro oscillator (8) arranged on the sample tube bracket (9), a sample magnetic reaction area arranged on the micro oscillator (8) and a sample tube (5) for connection;
the micro-fluidic chip (3) comprises a substrate layer (34) and a chip layer (33) arranged on the substrate layer (34), wherein the chip layer sequentially comprises a sample inlet (31), a single spiral type runner primary separation area, a magnetic separation secondary separation area and an outlet (32).
2. The integrated microfluidic separation device for circulating rare cells according to claim 1, wherein the holding carrier (4) comprises a chip base (41) including a magnet adapter (42) and an elastic clip (47), and a carrier upper cover (43) connected to the chip base (41), the carrier upper cover (43) has a soft plug socket (45) and an elastic clip (46) embedded in the carrier upper cover (43), the elastic clip (46) is engaged with the elastic clip (47) to close the carrier upper cover (43), the soft plug socket (45) is respectively engaged with the sample inlet (31) and the outlet (32) of the microfluidic chip; the movable magnet (6) is clamped into a magnet adapter (42) of the chip base (41) and corresponds to the magnetic separation secondary separation area of the microfluidic chip (3).
3. The integrated microfluidic separation device for circulating rare cells according to claim 1, wherein the sample magnetic reaction area comprises a sample inlet (51), a magnetic bead suspension inlet (52), a reaction chamber (21) and a sample line outlet (53), the sample line outlet (53) is connected to the sample line (5), and the sample inlet (51) and the magnetic bead suspension inlet (52) are communicated to the reaction chamber (21) through a microfluidic pump (1) connected to the sample line (5).
4. The circulating rare cell integrated microfluidic separation device according to claim 3, wherein the micro oscillator (8) comprises a sample magnetic reaction area connector (81) and a sample tube holder clamping groove (82), the sample magnetic reaction area connector (81) is connected with the fixed reaction cavity (21), a sample line outlet (53) is arranged on the sample magnetic reaction area connector (81), and the sample line outlet (53) is connected with the sample tube (5).
5. the circulating rare cell integrated microfluidic separation device according to claim 4, wherein the sample magnetic reaction area joint (81) is provided with an electromagnetic controller (83), the electromagnetic controller (83) comprises an electromagnet (833), an armature (831) matched with the electromagnet (833) and a spring (832) arranged between the electromagnet (833) and the armature (831), and the sample tube (5) at the sample line outlet (53) is clamped on the spring (832).
6. The circulating rare cell integrated microfluidic separation device of claim 4, wherein the sample tube rack clamping groove (82) is clamped into the sample tube rack (9), and the sample tube rack clamping groove (82) comprises two metal contacts (84) connected with two metal electrodes (91) in the sample tube rack (9).
7. the circulating rare cell-integrated microfluidic separation device according to claim 1, wherein the single-spiral-type flow channel primary separation region is composed of a single-spiral-type microchannel (10) having 4 or more complete spirals and at least 3 branches at the ends of the spiral-type flow channel.
8. the circulating rare cell-integrated microfluidic separation device according to claim 7, wherein the single-spiral micro flow channel (10) has a height of 40 to 110 μm, a channel width of 150 to 600 μm, and an inter-channel distance of 450 to 500 μm;
The chip layer sample inlet (31) is positioned in the middle of the single-spiral flow channel (10), and the distance from the first inner spiral radius of the single-spiral flow channel is 2.5-4.5 mm.
9. the integrated microfluidic separation device for circulating rare cells according to claim 1, wherein the magnetic separation secondary separation zone comprises 3 or more magnetic separation cells (35), each magnetic separation cell (35) is connected with a terminal branch of the spiral micro-channel (10) in the single-spiral flow channel primary separation zone, the outlet (32) is positioned at the tail end of the magnetic separation secondary separation zone, 3 or more outlets (32) correspond to the magnetic separation cells (35), and a corresponding number of cell collection tubes (7) are arranged at the outlets (32) for collecting CRCs and other cells.
10. A circulating rare cell integrated microfluidic separation method, characterized in that the circulating rare cell integrated microfluidic separation device of any one of claims 1 to 9 is adopted, and the method comprises the following steps:
1) Placing the micro-fluidic chip (3) into a clamping carrier (4);
2) Loading a sample magnetic reaction area on a sample magnetic reaction area connector (81) of a micro oscillator (8), connecting a sample tube bracket clamping groove (82) with a sample tube bracket (9), butting a metal electrode (91) on the sample tube bracket (9) with a metal contact (84) during connection, finally connecting a clamping carrier (4) and starting a power supply, starting the micro oscillator (8) to vibrate, attracting an armature (831) by an electromagnet (833) after the electromagnetic controller (83) is electrified, compressing a spring (832), clamping a sample tube (5) at a sample tube outlet (53) by the spring (832) to cut off fluid in the tube to prevent liquid which is not reacted in the sample magnetic reaction area from flowing out;
3) The method comprises the following steps that a monocyte layer (PBMCs) subjected to density gradient centrifugation treatment is added into a reaction cavity (21) of a sample magnetic reaction area through a sample inlet (51) by a micro-flow pump (1), meanwhile, a magnetic bead suspension is injected into the reaction cavity (21) through a magnetic bead suspension inlet (52), and the two are mixed and combined to react for 10-30 min;
4) The micro-oscillator (8) is closed, the electromagnet (833) of the electromagnetic controller (83) is powered off, the spring (832) of the electromagnetic controller (83) resets and rebounds, the sample tube (5) at the outlet (53) of the sample pipeline is opened, and the reaction completion liquid enters the microfluidic chip (3) through the outlet (53) of the sample pipeline to carry out cell separation;
5) The reaction finished liquid enters a single-spiral micro-flow channel (10) of a single-spiral flow channel primary separation area to be separated by fluid inertia force under the action of a micro-flow pump (1), cells with the cell diameter larger than 20 mu m separated by the fluid inertia force enter a first magnetic separation pool from an inner measuring flow channel, cells with the cell diameter of 15 +/-5 mu m and the medium particle size enter a second magnetic separation pool from a middle flow channel, cells with the cell diameter smaller than or equal to 7 mu m enter a third magnetic separation pool from an outer flow channel, under the action of a magnetic field of a moving magnet positioned below the magnetic separation pools, leukocytes connected with magnetic beads are gathered in each magnetic separation pool and separated from CRCs, the used moving magnet (6) is in a semi-ring shape, the leukocytes can also form semi-ring-shaped gathering, and the CRCs flow into a cell collection pipe (7) through an outlet (32) along a sample pipe (5) to be collected;
6) after the CRCs are collected, the microflow pump (1) is closed, the clamping carrier (4) is opened, the microflow control chip (3) is taken out, the movable magnet (6) in the chip base (41) of the clamping carrier is removed, the microflow control chip (3) which is taken out is put back into the clamping carrier (4), the microflow pump (1) is started, and the white blood cells connected with the magnetic beads are collected by a new cell collecting pipe (7).
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