CN108795693B

CN108795693B - Micro-fluidic chip for capturing rare cells in blood

Info

Publication number: CN108795693B
Application number: CN201810667851.XA
Authority: CN
Inventors: 孔祥菊; 石虎兵; 李燕雏; 徐琪
Original assignee: Chengdu Ruipukang Bio Tech Co ltd
Current assignee: Chengdu Ruipukang Bio Tech Co ltd
Priority date: 2018-06-26
Filing date: 2018-06-26
Publication date: 2021-01-01
Anticipated expiration: 2038-06-26
Also published as: CN108795693A

Abstract

The invention relates to biological and pathological body fluid detection, and relates to a microfluidic chip for separating high-purity rare cells from a whole blood cell population.

Description

Micro-fluidic chip for capturing rare cells in blood

Technical Field

The invention relates to biological and pathological body fluid detection, in particular to a microfluidic chip for separating high-purity rare cells from a whole blood cell population.

Background

Rare cells refer to some atypical cells in a fluid sample of an organism (including blood, pleural fluid, ascites, urine, cerebrospinal fluid, etc.). Research shows that the method has important guiding significance for collecting rare cells and utilizing the rare cells to complete NGS analysis and finding out potential disease treatment mechanisms, pathological mechanisms and targeted drug development. At present, the detection and research methods of rare cells in blood mainly comprise a flow sorting technology, a morphological separation method, a density gradient centrifugation method, a membrane filtration method and an immunomagnetic separation technology, for example, BD FACSAris can realize high-speed sorting of cells, but the sorted cells are damaged by instantaneous laser of flow sorting, and the activity of the cells is damaged after the sorting activity. Circulating Tumor Cells (CTCs) refer to tumor cells that have entered the peripheral blood of a human. Although the number of CTCs in peripheral blood of tumor patients is extremely small, it is generally about 10⁷CTCs in blood cells are only single-digit (2-10/ml) in magnitude, but CTCs are an extremely important tool for fluid biopsy, prognosis determination and inter-treatment follow-up. Because the number of circulating tumor cells is extremely rare, the requirements for the detection accuracy and specificity are high, and the further analysis of the circulating tumor cells is more difficult, the development of a portable method and a tool for separating the circulating tumor cells from the blood sample with high efficiency, high flux and high speed is urgently needed.

Circulating tumor cells in human peripheral blood are tumor cells (CTCs) or cell Clusters (CTM) which are disseminated into peripheral blood circulation from tumor lesions, and the surviving CTCs or CTM leave the blood circulation and enter local microenvironment of secondary organs, proliferate and grow under the action of various growth factors and finally form metastases. Circulating tumor cells are an important source of tumor blood vessel metastases, and distant metastasis is one of the direct causes of tumor treatment failure, recurrence and death, however, as long as early detection and intervention can greatly reduce recurrence and metastasis rates, detection of circulating tumor cells from blood is increasingly attracting attention; by capturing and analyzing CTCs, the kit can assist clinicians in tumor metastasis and recurrence prediction and early warning, perform evaluation of overall survival time (OS) and progression-free survival time (PFS) of patients, monitor curative effect of clinical radiotherapy and chemotherapy and the like, and further has important clinical significance in guiding individualized medical treatment and improving survival state of tumor patients.

However, a prerequisite for clinical diagnosis or laboratory analysis of CTCs is the availability of sufficient CTC cells. Since CTCs are present in peripheral blood every 10⁶～10⁷And each monocyte only contains 1 CTCs, so that extremely high requirements are put on the sensitivity and specificity of the CTCs detection technology. Various CTCs detection schemes mainly comprise a CTCs separation and enrichment system and a CTCs detection and identification system.

In recent years, a series of capture technologies of circulating tumor cells are developed by combining a microfluidic chip technology with antibody capture. The principle of sorting and enriching CTCs by microfluidic chips is mainly divided into 4 types: sorting by utilizing the affinity of antigen and antibody; sorting by using different physical characteristics of cells, such as the size and deformability of the cells and the mechanical properties of the cells with different sizes in a flow field; thirdly, sorting by using the magnetic and antibody-connecting function of the immunomagnetic beads; and fourthly, sorting and the like by utilizing the difference of the electrical properties of different cells. The method of the microfluidic cell immune chip is to detect the CTCs by a microfluidic device (micro-fluidic device), the detection method has the characteristics of extremely high specificity, sensitivity and the like, the captured CTCs have cell activity and can be separated and used for cell culture and other various downstream technical researches, and the method is a brand new and efficient method for researching the clinical application value of the CTCs. Currently, products such as antibody-dependent CTCs chips (On-Q-ity, Waltham, MA), ClearCell (TM) system based On size deformability principle (Clearbridge BioMedics, Singapore), IsoFluxTM system based On immunomagnetic beads (Fluxion), ApostreamTM (Apocell) based On hydrodynamic characteristics, and DEPArray (TM) system based On dielectrophoresis principle are being marketed. However, the microfluidic chip generally has the advantages of small sample amount, high sensitivity and the like, but because the amount of blood samples detected by each device is small, the selective shift of the samples is inevitable, and the occurrence rate of false negative is high, the microfluidic chip has important influence on sample analysis and clinical diagnosis. In addition, since the chip is usually small in size, when blood passes through, the flow rate is too small, the efficiency is too low, and the possibility of blood coagulation is increased, too large flow rate can result in short binding time to a target component, the capture is insufficient, and in addition, the shearing force to which blood cells are subjected is also large, and hemolysis can be caused, so that the development of a microfluidic chip detection system with higher flow rate, better binding efficiency and lower hemolysis becomes the focus of current research, and thus reliable data support can be provided for clinic. On the other hand, some special chips are expensive in manufacturing cost and inconvenient to popularize and apply. How to improve the above problems encountered in cell sorting by the microfluidic chip technology and fully exert the advantages thereof will be the key for the microfluidic chip (chip set) to perform the capture of CTCs.

The inventor of the invention provides a chip for efficiently collecting blood circulation tumor cells through creative work. The chip has the following advantages: the improved micro-fluidic chip adopts a specific spoiler and a spoiler column, so that the damage to blood cells is reduced while the high-efficiency collection of circulating tumor cells is guaranteed, and the risk of hemolysis is reduced; the circulating tumor cells captured by the microfluidic chip technology can be taken out without damage for analysis or in vitro culture, so that the effects of the circulating tumor cells on the aspects of tumor recurrence, drug resistance prediction and the like can be reflected; the chip of the present invention can be used not only for capturing rare cells in blood, but also for capturing various cells or cell components in other biological fluids.

Disclosure of Invention

In one aspect, the invention provides a microfluidic chip for capturing rare cells or cell components in biological fluid including blood, wherein a plurality of turbulence columns are arranged in the chip to form a turbulence column array, so that the contact area of the internal surface of the chip and the fluid is increased, and the probability of capturing target components is increased.

In one aspect, the present invention provides a method for designing a microfluidic chip, comprising: the principle of the method is that under the condition of meeting a certain blood flow, cells or other components in blood can have longer residence time in the chip, so that more target components can be combined under the condition of the same flow, and meanwhile, the higher flow rate can be adopted under the condition of combining the same amount of target components, thereby improving the working efficiency of the chip. In order to meet the principle, the design method comprises the step of arranging a turbulence column in a lane of the chip, and is characterized in that the turbulence column can not only generate the karman vortex street when fluid passes through the lane. Due to the existence of the karman vortex street, the stagnation time of the fluid in the flow channel in unit area is prolonged, and the fluid can be separated from the vortex street to continue flowing after being stagnated for a certain time, so that the flow path of the fluid in a lane and the stagnation time of the fluid are further increased, the local part around a turbulence column and the whole flow velocity of the fluid in a chip are influenced, the contact time of components in the fluid and the lane is prolonged, and the capture of target components is facilitated.

In one aspect, the present invention provides a microfluidic chip or chip set, the microfluidic chip including an array of spoiler bars inside, the spoiler bars enabling karman vortex streets to be generated when fluid passes through the array of spoiler bars.

According to any one of the preceding aspects, the component is a circulating rare cell, preferably a circulating tumor cell.

According to any one of the preceding aspects, wherein the micro-fluidic chip main body comprises a substrate layer and a cover layer which are sequentially arranged from bottom to top, a component capture chamber is arranged between the substrate layer and the cover layer, a fluid inlet and a fluid outlet which are communicated with the component capture chamber are arranged on the cover layer, the component capture chamber is divided into a buffer area and a lane part, a turbulence column array is arranged in the lane along the flow direction, and the inner surface of the component capture chamber and the surface of the turbulence column are both smooth transition curved surfaces.

According to any one of the previous aspects, the buffer area is provided with a front-end flow dividing block near the inlet and the outlet, which is a junction of lanes, and has the functions of dividing flow, reducing flow speed and buffering.

According to any of the preceding aspects, wherein the lane is divided into two or more lanes arranged in parallel by a dividing block, preferably each lane comprises 5-500 spoiler columns, 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 turbulence columns are arranged in parallel in the swimming channel at a specific pitch of 0.1 to 5, preferably 0.5 to 1, diameters (major or minor) of the turbulence columns in the same direction.

According to any of the preceding aspects, the height of the turbulence columns is 100-500 μm, the length of the columns is 1.0-3.25mm, the length of the columns is 0.5-1.0mm, each group of turbulence columns consists of 3 separate turbulence columns arranged in a delta shape, and the inter-group turbulence column spacing is 0.1-1mm, preferably 0.3-0.7mm, and most preferably 0.45 mm; the inter-group turbulence column spacing is 0.1-1.5mm, preferably 0.5-1mm, most preferably 0.7 mm.

According to any one of the preceding aspects, the component capture chamber inlet flow rate is from 0.01 to 0.05 m/s.

According to any one of the preceding aspects, two ends of the shunting block respectively form parts of an inlet and an outlet of a lane, the surfaces of the inlet and the outlet are subjected to anti-hemolysis treatment to form smooth transition curved surfaces, and optionally, the cross section of the inlet and the outlet can be circular arc, inverted circle, fillet rectangle, fillet trapezoid and the like.

According to any one of the preceding aspects, the surface of the spoiler column in the flow direction is a smooth transition curved surface, specifically, the cross section of the spoiler column is cylindrical, round rectangle, ellipse, dumbbell, streamline, spindle or olive shape, or is similar to the shape of two water drops in a tail-to-tail connection shape, preferably olive shape, so that the surface of the spoiler column in the flow direction is a smooth transition curved surface, thereby reducing the damage of mechanical shearing force to cells in a body.

According to any one of the preceding aspects, wherein the component capture chamber is loaded on its inner surface and the surface of the flow perturbation column with a capture ligand that binds to a target component in the fluid, preferably the capture ligand is a streptavidin-biotin-anti-EPCAM antibody complex.

According to any one of the preceding aspects, the material of the base sheet or the cover sheet is selected from silicon, glass, siliconized glass, PDMS, PMMA, or one or more than two high molecular polymer materials selected from polypropylene, cyclic olefin copolymer, cyclic olefin polymer, polymethyl methacrylate and polycarbonate.

In one aspect, the present invention provides a method for manufacturing a microfluidic chip according to any one of the preceding aspects: the method comprises the following steps:

1. manufacturing a chip mask;

2. preparing and exposing a photosensitive film: constructing a chip design pattern convex framework by using a photosensitive film, attaching a mask on the photosensitive film, and irradiating by using ultraviolet rays; removing the exposed dissolved photosensitive film, washing, and drying in an oven to obtain a convex framework of the chip design pattern for later use;

3. preparing a PDMS mold: stirring the SLYGARD 184 reagent uniformly at room temperature, pouring the mixture into a PDMS chip grinding tool which takes a chip design pattern convex framework as a bottom plate, vacuum degassing, drying in an oven, and solidifying the PDMS mould;

4. bonding PDMS: putting the PDMS chip mould and the glass slide carrier into a Plasma Cleaner (Plasma Cleaner) together for bonding to obtain a designed chip finished product;

5. chip coating: and putting the manufactured chip into the plasma cleaning machine again, taking out the chip after the chip is acted, and then injecting the target antibody into a chip flow channel for coating.

In one aspect, the invention also provides uses of the microfluidic chip of the invention, including for capturing rare components, preferably rare cells, more preferably circulating tumor cells, in a biological fluid, preferably blood.

Drawings

Fig. 1 is a schematic view of a fluid component capture system, wherein 11-15 are power plants, 2 are fluid conduits, 3 are cell capture devices, 4 are human bodies, and 5 are anticoagulant release devices.

Fig. 2 is a schematic view of a microfluidic chip and its connections.

FIG. 3 the haemolysis is detected spectrophotometrically.

FIG. 4 is a graph of circulating tumor cell collection.

FIG. 5 is a pressure simulation of the chip of the present invention. The design of the flow deflectors in the front of the buffer pool enables the flow in each swimming channel to be uniformly distributed, forms fluid retardation at the A/B/C position, and plays a role in controlling the flow in the flow channel.

FIG. 6 is a graph showing the simulation of the flow velocity of karman vortex street in the rear of the spoiler column, wherein the left graph shows the simulation of the flow velocity of karman vortex street, the right graph shows the simulation of the flow velocity of karman vortex street, and the arrow of the right graph shows the generation of karman vortex street.

FIG. 7 is a graph of circulating tumor cell collection.

Detailed Description

As used herein, "rare cells refer to some atypical cells in a fluid sample of an organism (including blood, pleural fluid, ascites, urine, cerebrospinal fluid, etc.). Examples of rare cells include, but are not limited to, Circulating Tumor Cells (CTCs), Circulating Endothelial Cells (CECs), and Circulating Multiple Myeloma Cells (CMMCs). Preferred rare cells are CTCs and CECs, and particularly preferred rare cells are CTCs. By "circulating tumor cells" (CTCs) is meant malignant tumor 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.

By "blood component" is meant 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, for example, due to current or past pregnancy, organ transplantation or infection.

"fluid" or "biological fluid" is intended to include natural fluids (e.g., blood, lymph, cerebrospinal fluid, urine, cervical lavage, saliva, and water samples), portions of these fluids, and fluids into which cells have been introduced (e.g., culture media and liquid tissue samples). The term also includes lysates.

The "capture unit" or "capture ligand" may, as the case may be, refer to a chemical sample for binding an analyte, or a component binding substance of its surface on which whole cells are to be relied upon. 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.

"channel" or "lane" refers to a gap through which fluid can flow. The channels may be capillaries on a hydrophobic surface, conduits, or hydrophilic textures of a hydrophobic surface to which an aqueous liquid may be confined.

By "fraction" of cells is meant any fraction that can be separated in a lysis solution. The cellular component can be an organelle (e.g., a nucleus, a juxta-nuclear compartment, a nuclear membrane, a mitochondrion, a chloroplast or a cell membrane), a polymer or molecular complex (e.g., a lipid, a polysaccharide, a protein (membrane, transmembrane or cytoplasm), a nucleic acid (native, therapeutic or pathogenic), a virion, or a ribosome), or other molecule (e.g., a hormone, ion, cofactor, or drug). By "fraction" of a cellular sample is meant a subset of cellular fractions contained in the sample.

By "enriched sample" is meant a sample containing such analytes: which has been treated to increase the relative amount of analyte relative to the sample normally present. For example, the sample may be enriched by increasing the amount of the 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 fold.

"Cross-section" refers to a profile side view image.

A "rare amount" of cells refers to less than 100 cells/ml of fluid, less than 10 cells/ml of fluid, or even less than 1 cell/ml of fluid.

Other features and advantages will be apparent from the following description and claims.

In order to achieve the object of the invention, the present inventors have devised a microfluidic chip device for rare cell capture through creative efforts; the cell capture device is connected in series or in parallel with the fluid conduit such that fluid can pass through the cell capture device to capture cells contained in the fluid. Preferably, the fluid is blood. Preferably, the cell is a circulating rare cell, further preferably, the cell is a circulating tumor cell. The cell capture device comprises a microfluidic chip or chip set.

In one embodiment, the invention provides a microfluidic chip for capturing rare cells or cell components in biological fluid including blood, wherein the chip is internally provided with a plurality of turbulence columns to form a turbulence column array.

In one embodiment, the design method of the microfluidic chip of the invention comprises arranging a turbulence column in a lane of the chip, wherein the turbulence column enables a fluid to generate a karman vortex street in the lane.

In one embodiment, the flow and flow distribution are simulated using Gambit2.4 version and ANSYS Fluent 19.0 version software to determine if the chip can generate a Karman vortex street.

In one embodiment, the chip main body comprises a substrate layer and a cover plate layer which are sequentially arranged from bottom to top, a component capture chamber is arranged between the substrate layer and the cover plate layer, a fluid inlet and a fluid outlet which are communicated with the component capture chamber are arranged on the cover plate layer, the component capture chamber is divided into a buffer area and a lane part, the buffer area is close to the inlet and the outlet of the component capture chamber and has the functions of reducing the flow rate and buffering for the junction of lanes, the lane part comprises one or more lanes, preferably the lane part is divided into at least two lanes by a flow dividing block, and a flow disturbing column array is arranged in each lane in the flow direction.

In one embodiment, the two ends of the shunting block form parts of an inlet and an outlet of the lane, respectively, and the surfaces of the inlet and the outlet are processed into smoothly-transiting curved surfaces through hemolysis prevention treatment, for example, the cross section of the inlet and the outlet can be circular arc, inverted circle, rounded rectangle, rounded trapezoid, etc.

In one embodiment, the diverter block is comprised of a front T-shaped baffle, a middle linear baffle, and a rear circular baffle, the front and rear baffles compressing the lane width such that the lane entrance and exit are narrower than the lane width, the middle baffle dividing the lane into a plurality of parallel lanes.

In one embodiment, the front end T-shaped guide plate is in a square hammer shape, the long diameter of the square hammer shape is 1.0-2.0mm, and the short diameter is 0.5-1.0 mm; the rear circular guide plate has a diameter of 1-3mm, preferably 2.4 mm.

In one embodiment, the cross section of the turbulence column is cylindrical, round rectangle, ellipse, dumbbell, streamline, spindle or olive shape, or the shape similar to the tail-to-tail connection of two water drops, preferably olive shape, so that the surface of the turbulence column in the flowing direction is a smoothly-transiting curved surface, thereby reducing the damage of mechanical shearing force to cells in the body. The height of the turbulence column array is equal to the internal height of the chip component capture chamber.

In one embodiment, the turbulator posts are disposed on a side of the cover plate adjacent to the base plate.

In one embodiment, the turbulator posts are disposed on a side of the base sheet adjacent to the cover sheet.

In one embodiment, each lane has a diameter of 3mm to 5mm, a height of 50 to 500 μm, a space between flow-disturbing columns of 0.1 to 5, preferably 0.5 to 1, major or minor diameters, a flow-disturbing column array height of 10 to 500 μm, and 5 to 500 flow-disturbing columns contained in each lane, forming a microarray.

In one embodiment, the height of the turbulence columns is 100-500 μm, the length of the columns is 1.0-3.25mm, the length of the columns is 0.5-1.0mm, each group of turbulence columns is composed of 3 independent turbulence columns arranged in a delta shape, the distance between the turbulence columns among groups is 0.1-1mm, preferably 0.3-0.7mm, and most preferably 0.45 mm; the inter-group turbulence column spacing is 0.1-1.5mm, preferably 0.5-1mm, most preferably 0.7 mm.

In one embodiment, the component capture chamber inlet flow rate is from 0.01 to 0.05 m/s.

In one embodiment, the lanes are arranged in parallel.

In one embodiment, the length of the substrate is 5 to 100mm, preferably 20 to 80mm, further preferably 30 to 60mm, most preferably 50 mm; the width is 5-50mm, preferably 10-30mm, most preferably 20 mm.

In one embodiment, the number of lanes is 1-20, preferably 4-15, further preferably 6-10, most preferably 8.

In one embodiment, the length of the lanes is 5-100mm, preferably 20-40mm, most preferably 30 mm; a width of 0.1 to 50mm, preferably 0.5 to 5mm, further preferably 1 to 3mm, most preferably 1.5 mm; the height is 0.05-0.5mm, preferably 0.05-0.1 mm.

In one embodiment, the material of the substrate or the cover sheet is silicon, glass, siliconized glass, PDMS, PMMA, etc., and may also be one or more than two high molecular polymer materials selected from polypropylene, cyclic olefin copolymer, cyclic olefin polymer, polymethyl methacrylate and polycarbonate.

In one embodiment, the cell capture device comprises a chip combiner, which is a medical grade connecting tube and a card slot, for holding a microfluidic chip, such that a plurality of chips are connected in parallel or in series into a chip set.

In one embodiment, the inner surface of the component capture chamber and the surface of the spoiler column are loaded with Streptavidin (Streptavidin) that is capable of specifically binding to labeled Biotin in an anti-epithelial cell adhesion molecule (EPCAM) antibody-Biotin (Biotin) complex, wherein the EPCAM antibody in the EPCAM antibody-Biotin complex is capable of specifically binding to EPCAM antigen on the surface of circulating tumor cells; wherein the linkage of the streptavidin-biotin complex in the streptavidin-biotin-EPCAM antibody complex is such that it competes for elution by high concentrations of biotin. Optionally, the inner surface of the component capture chamber and the surface of the spoiler column may also be coated with other capture ligands, such as antigens, antibodies, protein a, protein G, lectins, and the like.

In one embodiment, the present invention provides a method of making a microfluidic chip of the present invention: the method comprises the following steps:

1. manufacturing a chip mask;

4. bonding PDMS: putting the PDMS chip mold and the glass slide carrier into a plasma cleaning machine together for bonding to obtain a designed chip finished product;

5. chip coating: and putting the manufactured chip into a Plasma Cleaner (Plasma Cleaner) again, taking out the chip after the chip is acted, and then injecting the target antibody into a chip flow channel for coating.

In one embodiment, the mask size is (5-10) × (2-4) cm.

In one embodiment, the photosensitive film is 3 to 13 photosensitive films having a thickness of 35 μm/sheet.

In one embodiment, the photosensitive film making and exposing step comprises: constructing a convex framework of a chip design pattern by using a photosensitive film with the thickness of 35 mu m/piece specification, conventionally using 3-13 photosensitive films, pasting a mask on the photosensitive film, and irradiating for 30-120 seconds by using ultraviolet rays for exposure; carefully removing the exposed dissolved photosensitive film by using a cleaning solution and a soft brush, uniformly oscillating by using an oscillator in the cleaning process to ensure that the residual dissolved photosensitive film is fully removed, and washing by using deionized water; then putting the chip into an oven to be dried for 10-30 minutes to obtain a chip design pattern convex framework; observing the condition of the chip framework under a stereoscopic microscope to ensure that the edge of the chip framework is smooth and the micro turbulence column is not damaged; the skeleton finished product is sealed and stored by a dust-free self-sealing bag.

In one embodiment, the fabricating the PDMS mold includes: the SLYGARD 184 reagent is mixed according to the mass ratio of 1: stirring uniformly at room temperature in a proportion of 10, slowly pouring into a PDMS chip grinding tool taking a convex framework of a chip design pattern as a base plate, and vacuum degassing for 8-15 minutes by using a vacuum pump (with the pressure of 0.06 pa). And (4) after degassing is finished, drying in an oven at 60 ℃ (3 hours) until the PDMS mold is solidified.

In one embodiment, the bonded PDMS: putting the PDMS chip mold and the glass slide carrier into a plasma cleaning machine together for bonding; vacuumizing for 2 minutes, and pressing PDMS on the glass slide; and then heating the mixture on a flat plate at the temperature of 60 ℃ for 30 minutes to obtain a finished product of the designed chip (the finished product is sealed and stored by a dust-free self-sealing bag).

In one embodiment, the chip coating comprises putting the fabricated chip into a Plasma Cleaner (Plasma Cleaner) again, taking out the chip after 1-5 minutes, preferably 2 minutes; slowly injecting the target antibody into the chip flow channel, and keeping the target antibody in an environment at about 4 ℃ for 1-20 hours, preferably 12 hours; the antibody is removed and 5% BSA is added to block for 1-5 hours, preferably 2 hours, at about 37 deg.C.

The microfluidic chip set disclosed by the disclosure is connected to an extracorporeal circulation system (as shown in fig. 1) in a serial or parallel mode according to the actual blood sampling quantity requirement. The blood is guided by the blood vessel and enters a circulation power system of an extracorporeal circulation system to obtain extracorporeal circulation power, and the blood is branched into one or more parallel catheters at the far end, and the catheters are respectively connected into the microfluidic chip set and the bypass, and a flow limiting valve is arranged after entering the microfluidic chip set. The connection is shown in fig. 2. Preferably, the flow limiting valve is a three-way flow limiting valve. Through setting up the bypass pipeline for can conveniently adjust the velocity of flow and the pressure through the component capture room, be difficult to produce higher shearing force to the blood cell, prevent the hemolysis, and be difficult to cause the pipeline to block up.

The micro-fluidic chip or chip set is connected to a blood circulation system, circulating blood passes through the micro-fluidic chip micro-channel after being mixed and contacts with the antibody loaded on the surface, and circulating tumor cells are captured from a blood sample and fixed on the surface of the micro-channel by utilizing the combination of the antibody loaded on the surface of the micro-channel and the specific antigen on the surface of the circulating tumor cells.

In one embodiment, the release of the antibody and the circulating tumor cells captured by the antibody from the surface of the microchannel is achieved by cutting off the streptavidin-biotin complex, thereby obtaining high-purity circulating tumor cells, and the collection of the captured circulating tumor cells is achieved by washing the microfluidic chip with a cell washing solution after the end of the circulating collection. The cell washing solution is a buffer solution containing high-concentration biotin protein, and a preservative can be added according to the preservation requirement. The eluted CTC cells were stained with CD45 and CK8/18 fluorescent antibodies, and the population of CD45(-), CK8/18(+) cells were judged as CTC cells by observation with a fluorescent microscope.

The anticoagulated blood tested by spectrophotometry according to the present disclosure was tested for hemolysis of erythrocytes by the chip of the present disclosure (fig. 3).

Compared with the prior art, the invention has the following advantages and characteristics:

1. compared with a circulating tumor cell capturing method based on targeted polypeptide and a microfluidic chip, the method does not need to perform erythrocyte lysis before CTC collection, and instead, the method protects erythrocytes in the CTC collection process; 2. the combination of the streptavidin-biotin compound can be interrupted by a cell cleaning solution, and the circulating tumor cells captured by the microfluidic chip technology can be taken out without damage for further analysis or in-vitro culture, so that the effect of the circulating tumor cells can be better exerted. 3. The inlet and the outlet of the lane of the microfluidic chip set and the inlet and the outlet of the connecting pipeline are both designed to be in a smooth transition curve shape, so that potential damage to blood cells caused by the internal structure of the system is avoided; 4. turbulence column microarray has both increased the surface area with blood contact and has prevented the mechanical damage to the blood cell again, compares current CTC and detects or blood cell clearing device, and the turbulence column chip of this patent design more is suitable for online blood collection system to carry out blood collection guarantee CTC high-efficient collection with the chipset form, and reduced the blood and dissolved the blood phenomenon and taken place, ensured the quality of blood after the circulation. 5. The chip of the invention not only can capture rare cells in an online extracorporeal circulation manner, but also can realize the extracorporeal circulation of fluid in an offline manner, thereby improving the capture efficiency. 6. The chip of the present invention can be used not only for capturing rare cells in blood, but also for capturing various cells, or cell components, in other biological fluids.

Examples

The invention is further illustrated by the following examples.

Example 1: fabrication of chips

1. Mask manufacturing: and outputting a chip mask by using a high-precision laser printer, wherein the mask is 5-10 multiplied by 2-4cm in size.

2. Preparing and exposing a photosensitive film: constructing a chip design pattern convex framework by using a photosensitive film with the thickness of 35 mu m/piece, conventionally using 3-13 photosensitive films, pasting a mask on the photosensitive film, and irradiating for 30-120 seconds by using ultraviolet rays for exposure; carefully removing the exposed dissolved photosensitive film by using cleaning solution and a soft brush, uniformly oscillating by using an oscillator in the cleaning process to ensure that the residual dissolved photosensitive film is sufficiently removed, and washing for 3 times by using RO clear water; then putting the chip into an oven to be dried for 10-30 minutes to obtain a chip design pattern convex framework; observing the condition of the chip framework under a stereoscopic microscope to ensure that the edge of the chip framework is smooth and the micro turbulence column is not damaged; the skeleton finished product is sealed and stored by a dust-free self-sealing bag.

3. Preparing a PDMS mold: the SLYGARD 184 reagent is mixed according to the mass ratio of 1: stirring uniformly at room temperature according to the proportion of 10, slowly pouring into a PDMS chip grinding tool taking a convex framework of the chip design pattern as a bottom plate, and vacuumizing for 8 minutes by using a vacuum pump (with the pressure of 0.06 pa). And (4) after degassing is finished, drying in an oven at 60 ℃ (3 hours) until the PDMS mold is solidified.

4. Bonding PDMS: putting the PDMS chip mould and the glass slide carrier into a Plasma Cleaner (Plasma Cleaner) together for bonding; vacuumizing for 2 minutes, and pressing PDMS on the glass slide; and then heating the mixture on a flat plate at the temperature of 60 ℃ for 30 minutes to obtain a finished product of the designed chip (the finished product is sealed and stored by a dust-free self-sealing bag). The chip layout shown is shown in fig. 4.

5. Chip coating:

(1) the chip was washed 3 times with 200. mu.L sterile PBS to confirm that no air bubbles remained in the chip channels.

100ul of streptavidin was injected into the chip with a syringe, and then the chip was placed in a wet box and coated at 37 ℃ for 2 hours.

(2) Coating of capture antibodies

2.1 the human EpCAM capture antibody (biotin label) was added to a 1 XPBS sterile solution and mixed to prepare an antibody capture working solution with a concentration of 5. mu.g/ml.

2.2 mu.L of the antibody capture working solution was injected into the chip with a syringe, and then the chip was placed in a wet box and incubated overnight at 4 ℃. And ensuring that all chip flow channels are always covered with the antibody capture working solution in the whole incubation process.

2.3 the chip coated with the capture antibody was washed 3 times with 200. mu.L of 1 XPBS sterile solution to remove the working solution of the capture antibody remaining on the chip.

2.4 blocking the chip with 200. mu.l of 5% BSA solution at 27 ℃ for 2 hours.

2.5 at this point, the coating of the capture antibody on the chip is complete and ready for use.

Example 2: functional parameter detection of a chip

A grid model of the chip design was created in the gambit version 2.4 software and the flow regime of the liquid fluid in the chip was simulated using ANSYS flow version 19.0 software. As can be seen from simulation data, the flow of the fluid in each flow channel is uniformly distributed, and the T-shaped guide plate uniformly guides the liquid in the buffer tank to each flow channel; secondly, simulating the total flow rate when the inlet flow rate is set to be 0.01m/s-0.05m/s, setting the fluid density to be 'WATER-LIQUID', and calculating to obtain the flow rate in each flow channel to be 0.026-0.199 m/s; and a karman vortex street is formed behind each turbulence column (as shown in figures 5 and 6). The design requirement is met.

Example 3: capture capability detection of breast cancer circulating tumor cells

The chip prepared in example 1 was inserted into a blood container containing 10ml of breast cancer patient blood. The blood is guided out by the blood vessel and then enters the extracorporeal blood circulation system comprising the microfluidic chip set.

The micro-fluidic chip group formed by connecting the medical catheter in parallel/series is connected, and the anticoagulant sustained-release amplifier at the front end is opened.

And turning on a power switch, and continuously and circularly collecting the cell suspension for 1 hour.

And closing a power switch of the device, closing the automatic front-end anticoagulant sustained-release dispenser, and finishing the collection of the circulating tumor cells.

The wash chip was injected 3 times with 200 μ L of 1 XPBS sterile solution.

Observation of circulating tumor cells and hemolysis

The haemolysis of erythrocytes in the blood after circulation was detected by spectrophotometry, and no abnormal increase in the hemoglobin value was observed (FIG. 3).

Observing and collecting the number of circulating tumor cells under a mirror; CTC cells were judged by cell morphology size and nuclear to cytoplasmic ratio (fig. 7A).

Example 4: breast cancer cell capture efficiency detection

The wash chip was injected 3 times with 200 μ L of 1 XPBS sterile solution.

Injecting 200 μ l cell cleaning solution (containing high concentration biotin) into the chip with a syringe, standing at room temperature for 30min to separate streptavidin-biotin complex, and releasing biotin-EPCAM complex.

The chip was washed 3 times with 200. mu.L of 1 Xsterile PBS and the cell wash was collected.

Collected circulating tumor cells were fixed for 30 minutes by taking 200. mu.l of 95% ethanol with a syringe.

The residual detection antibody mixture was removed by washing 3 times with 200. mu.L of 1 Xsterile PBS.

CTC cells were stained with CK8 and CD45 for fluorescence and visualized under a fluorescent microscope. CK8(+) and CD45(-) indicate that the cells are tumor cells, demonstrating device capture efficiency, the results are shown in fig. 7B.

The result shows that the invention can be used for detecting the circulating tumor cells in the peripheral blood of a breast cancer patient, the circulating tumor cell detection sensitivity of the breast cancer is improved by scanning the blood as much as possible, and particularly, the detection sensitivity of the circulating tumor cells of the breast cancer is obviously improved by using a micro-fluidic chip set which is innovatively designed. Meanwhile, on the basis of scientific research field and clinical test verification, the circulating tumor cells of epithelial origin in peripheral blood of breast cancer patients are effectively detected by a method of identifying specific cytokeratin CK, leukocyte common antigen CD45 and cell nucleus of the circulating tumor cells by immunity and adding cell morphological staining.

The present invention is not limited to the particular embodiments shown and described herein, but various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the specification.

Claims

1. The utility model provides a micro-fluidic chip of component in seizure biological fluid, wherein the chip main part includes substrate layer and the cover plate layer that sets gradually from bottom to top, be equipped with the component capture room between substrate layer and the cover plate layer, be equipped with on the cover plate layer with fluid inlet and fluid outlet that the component capture room communicates, the component capture room divide into buffer and lane part, the lane passes through the reposition of redundant personnel piece and divide into two or more than two parallel arrangement's lane, be provided with the vortex column array along the flow direction in the lane, the vortex column is according to specific interval parallel arrangement in the lane, the interval is the diameter of 0.1-5 vortex columns, the vortex column makes the fluid produce karman vortex street when passing through in the chip, wherein the inside corner surface of micro-fluidic chip is smooth transition's curved surface.

2. The microfluidic chip according to claim 1, wherein each lane contains 5-500 turbulence columns.

3. The microfluidic chip according to claim 2, wherein each lane contains 15-100 turbulence columns.

4. The microfluidic chip according to claim 3, wherein 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 turbulence columns are contained in each lane.

5. The microfluidic chip according to claim 4, wherein each lane contains 17 turbulence columns.

6. The microfluidic chip according to claim 5, wherein the pitch is 0.5 to 1 diameter of the turbulence column.

7. The microfluidic chip according to any of claims 1 to 6, wherein the two ends of the shunting block respectively form parts of an inlet and an outlet of a lane, and the surfaces of the inlet and the outlet are processed by anti-hemolysis treatment to be smooth-transition curved surfaces.

8. The microfluidic chip according to claim 7, wherein the inlet and outlet have a cross-section selected from the group consisting of circular arc, rounded circle, rounded rectangle, and rounded trapezoid.

9. The microfluidic chip according to any of claims 1 to 6, wherein the surface of the flow perturbation column in the flow direction is a smoothly transitioning curved surface.

10. The microfluidic chip according to any of claims 1 to 6, wherein the cross-section of the turbulator is cylindrical, rectangular with rounded corners, oval, dumbbell, streamline, spindle or olive shaped, or two water droplets in a tail-to-tail shape.

11. The microfluidic chip according to any of claims 1 to 6, wherein the cross-section of the flow perturbation column is olive-shaped.

12. The microfluidic chip according to any of claims 1 to 6, wherein the inner surface of the component capture chamber and the surface of the flow perturbation column are loaded with capture ligands that bind to target components in the fluid.

13. The microfluidic chip according to claim 12, wherein the capture ligand is a streptavidin-biotin-anti-EPCAM antibody complex.

14. The microfluidic chip according to any of claims 1 to 6, wherein the substrate or the cover is made of a material selected from silicon, glass, siliconized glass, PDMS, PMMA, or a high molecular polymer material selected from one or two of polypropylene, cyclic olefin copolymer, cyclic olefin polymer, polymethyl methacrylate and polycarbonate.

15. Use of a microfluidic chip according to any of claims 1 to 6, characterized in that it is used for capturing circulating rare cells.

16. The use according to claim 15, wherein the circulating rare cells are circulating tumor cells.

17. A method of designing a microfluidic chip according to any one of claims 1 to 14, the method comprising providing turbulence columns in the lanes of the chip, wherein the turbulence columns cause karman vortex streets to form in the fluid as it passes through the lanes.

18. A method of making a microfluidic chip according to any of claims 1-14, comprising: the method comprises the following steps:

(1) manufacturing a chip mask;

(2) and preparing and exposing a photosensitive film: constructing a chip design pattern convex framework by using a photosensitive film, pasting a mask on the photosensitive film, and irradiating by using ultraviolet rays; removing the exposed dissolved photosensitive film, washing, and drying in an oven to obtain a convex framework of the chip design pattern for later use;

(3) and manufacturing a PDMS mold: stirring the SLYGARD 184 reagent uniformly at room temperature, pouring the mixture into a PDMS chip grinding tool which takes a chip design pattern convex framework as a bottom plate, vacuum degassing, drying in an oven, and solidifying the PDMS mould;

(4) bonding PDMS: putting the PDMS chip mold and the glass slide carrier into a plasma cleaning machine together for bonding to obtain a designed chip finished product;

(5) and chip coating: and putting the manufactured chip into the plasma cleaning machine again, taking out the chip after the chip is acted, and then injecting the target antibody into a chip flow channel for coating.