Magnetic field-fluid field synergistic micro-fluidic chip and application thereof

Abstract

The invention discloses a magnetic field-fluid field synergistic micro-fluidic chip and application thereof. The magnetic field-fluid field synergistic micro-fluidic chip comprises a top inlet and outlet layer 1, a middle micro-fluidic channel layer 2, a bottom substrate layer 3, a sample inlet 4, a buffer solution inlet 5, a sample outlet 6, a waste liquid outlet 7, a sample input flow channel 8, a buffer solution input flow channel 9, a waste liquid output flow channel, a main body flow channel 10, a waste liquid output flow channel 11, a sample output flow channel 12, an empty window 13 and a magnet 14. According to the micro-fluidic chip and the related detection system, circulating tumor cell suspension with extremely low content can be rapidly, simply and conveniently separated from a cancer patient blood sample with complex components, and the obtained cell suspension is high in purity and clean in background and can be used for subsequent downstream analysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic chips and relates to a magnetic field-fluid field coordinated microfluidic chip and applications thereof. Background Art

[0002] In recent years, cancer has become one of the leading causes of death worldwide, with its morbidity and mortality rates continuing to rise. Tumor metastasis is the primary cause of its high mortality rate. Circulating tumor cells (CTCs), tumor cells that shed from the primary tumor and enter the bloodstream, have been shown to play a key role in distant tumor metastasis. Numerous studies have demonstrated that the number and molecular characteristics of CTCs can be used to assess tumor progression, evaluate treatment efficacy, and monitor prognosis, possessing significant clinical and research value.

[0003] However, since the concentration of circulating tumor cells in peripheral blood is extremely low (usually 1-10 CTCs per ml of blood, and the number of white blood cells is around 10 6 Detection and enrichment of these cells have always been challenging. Traditional enrichment methods, such as immunomagnetic capture, cell size screening, or gradient centrifugation, can achieve initial sorting to a certain extent, but they suffer from long processing times, cumbersome procedures, damage to cell viability, and low enrichment purity, making them difficult to meet the clinical needs of high efficiency, automation, and high throughput.

[0004] With the rapid development of microfluidic technology, it has shown significant advantages in the field of CTC sorting. Through the precise design of parameters such as microchannel structure, fluid shear force, magnetic field distribution, and electric field effect, microfluidic chips can achieve non-labeling, high-throughput, and low-damage capture of rare circulating tumor cells, and adapt to subsequent operations at the single-cell level. However, existing microfluidic platforms still have multiple limitations, such as high structural complexity, difficulty in achieving batch packaging and convenient replacement, and the presence of a large number of non-target cells in the enriched cells. In addition, existing technologies often cause damage to cell structure or loss of activity in the process of capturing, sorting, and purifying CTCs, making it difficult to meet the needs of downstream analysis such as subsequent RNA sequencing and gene mutation detection.

[0005] Currently, existing technologies are unable to achieve rapid enrichment of circulating tumor cells in the blood and efficient removal of background cells, and are unable to simultaneously provide a complete process for producing a circulating tumor cell suspension with high purity and high activity.

[0006] Therefore, there is an urgent need to provide a microfluidic chip for high-throughput and efficient sorting and enrichment of circulating tumor cells in the blood of cancer patients. Summary of the Invention

[0007] In response to the shortcomings of existing technologies and actual needs, the present invention provides a magnetic field-fluid field synergistic microfluidic chip and its application, which can quickly and easily separate extremely rare circulating tumor cell suspensions from complex blood samples of cancer patients. The obtained cell suspension has high purity and clean background, and can be used for subsequent downstream analysis.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a microfluidic chip, comprising: a three-layer structure stacked together and sealed together, with an embedded permanent magnet, the three layers comprising, from top to bottom, a top inlet and outlet layer 1, a middle microfluidic channel layer 2, and a bottom base layer 3, each layer having a rectangular window 13 in the middle;

[0010] The top inlet and outlet layer 1 is provided with a sample inlet 4, a buffer inlet 5, a sample outlet 6, and two waste liquid outlets 7; the top inlet and outlet layer 1 is connected to the middle microfluidic channel layer 2, and each inlet and outlet is connected to the corresponding inlet and outlet of each branch flow channel on the channel layer;

[0011] The middle microfluidic channel layer 2 is provided with two parallel main flow channels 10, two sample input flow channels 8, a buffer input flow channel 9, two waste liquid output flow channels 11 and two sample output flow channels 12;

[0012] Among them, the sample inlet 4 is connected to the sample input flow channel 8, and is split at the sample inlet 4, and is respectively merged into the two main flow channels 10 from both sides, and the flow channels are axially symmetrically distributed; the buffer inlet 5 is connected to the buffer input flow channel 9, and is split at the buffer inlet 5, and is respectively merged into the two main flow channels 10 from both sides, and the flow channels are axially symmetrically distributed; the waste liquid outlet 7 is connected to the waste liquid output flow channel 11, and flows from the main flow channels 10 on both sides into the waste liquid output flow channels 11 and the waste liquid outlet 7 on both sides respectively; the sample outlet 6 is connected to the sample output flow channel 12, and flows from the main flow channels 10 on both sides into the two sample output flow channels 12 in the middle, and the two sample output flow channels 12 converge at the sample outlet 6; wherein, the sample input flow channel 8 and the waste liquid output flow channel 11 are located on the outside of the overall flow channel, the buffer input flow channel 9 and the sample output flow channel 12 are located on the inside of the overall flow channel, and the entire structure is axially symmetrically distributed;

[0013] The bottom base layer 3 is a smooth, flat bottom plate; the rectangular window 13 is a hollow part opened in the middle of the main flow channel 10 after the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom base layer 3 are assembled, and the rectangular window 13 has a magnet 14 embedded in it.

[0014] The microfluidic chip of the present invention can quickly and easily separate extremely rare circulating tumor cell suspensions from complex blood samples of cancer patients, and the obtained cell suspensions have high purity and clean background, and can be used for subsequent downstream analysis.

[0015] Figure 1 Schematic diagram of the structure of the microfluidic chip of the present invention, as shown in Figure 1 As shown, the sample solution is input from the sample inlet 4, enters the sample input flow channel 8, first flows horizontally to both sides, then flows longitudinally through the arc-shaped flow channel, and finally enters the main flow channel 10. The buffer solution (such as PBS solution) is input from the buffer inlet 5, enters the buffer input flow channel 9, first flows horizontally to both sides, then flows longitudinally through the arc-shaped flow channel, and finally enters the main flow channel 10.

[0016] In the present invention, the bottom substrate layer 3 is a smooth and flat bottom plate with a thickness of 100-2000 μm (eg, 100 μm, 1000 μm, 1500 μm, 1800 μm or 2000 μm).

[0017] In the present invention, the rectangular window 13 is a hollow portion opened in the middle of the main flow channel 10 after the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom base layer 3 are assembled, for accommodating the inner magnet 14.

[0018] Preferably, the diameter of the five entrances and exits of the top entrance and exit layer 1 is 1000-4000 μm (for example, 1000 μm, 2000 μm, 3000 μm or 4000 μm); the thickness of the top entrance and exit layer 1 is 900-4500 μm (for example, 900 μm, 1000 μm, 3000 μm, 4000 μm or 4500 μm).

[0019] Preferably, the channel depth of the middle microfluidic channel layer 2 is 50-400 μm (eg, 50 μm, 100 μm, 300 μm, 350 μm or 400 μm).

[0020] Preferably, the main flow channels 10 on both sides of the middle microfluidic channel layer 2 have a width of 750 to 3000 μm (for example, 750 μm, 1000 μm, 2000 μm or 3000 μm) and a length of 35 to 150 mm (for example, 35 mm, 50 mm, 100 mm or 150 mm), and the inner channel walls of the main flow channels 10 on both sides are 30 to 55 mm (for example, 30 mm, 35 mm, 40 mm, 50 mm or 55 mm) apart and are distributed in parallel and symmetrical manner.

[0021] Preferably, the sample input flow channel 8 has a flow channel width of 250 to 1000 μm (e.g., 250 μm, 300 μm, 500 μm, 800 μm, or 1000 μm), a height of 10 to 30 mm (e.g., 10 mm, 20 mm, or 30 mm), and a width of 35 to 60 mm (e.g., 35 mm, 40 mm, 50 mm, or 60 mm). The inner radius of the arc-shaped flow channel of the sample input flow channel 8 is 3 to 10 mm (e.g., 3 mm, 5 mm, 8 mm, or 10 mm). The flow channel width of the buffer input flow channel 9 is 10 to 30 mm (e.g., 10 mm, 20 mm, or 30 mm). The thickness is 400-1600 μm (for example, 400 μm, 600 μm, 800 μm, 1000 μm, 1500 μm or 1600 μm), the height is 5-25 mm (for example, 5 mm, 10 mm, 15 mm, 20 mm or 25 mm), the width is 34.3-57.2 mm (for example, 34.3 mm, 40 mm, 45 mm, 50 mm or 57.2 mm), and the inner radius of the arc-shaped flow channel of the buffer input channel 9 is 5-20 mm (for example, 5 mm, 10 mm, 15 mm or 20 mm).

[0022] Preferably, the shortest distance between the sample input channel 8 and the buffer input channel 9 at the confluence in the main flow channel 10 is 100-400 μm (eg, 100 μm, 200 μm, 300 μm or 400 μm).

[0023] Preferably, the waste liquid output channel 11 has a channel width of 350 to 1400 μm (for example, 350 μm, 400 μm, 800 μm, 1000 μm, 1200 μm or 1400 μm) and a height of 20 to 85 mm (for example, 20 mm, 40 mm, 60 mm, 80 mm or 85 mm).

[0024] Preferably, the sample output channel 12 has a channel width of 400 to 1600 μm (for example, 400 μm, 800 μm, 1000 μm or 1600 μm) and a height of 20 to 85 mm (for example, 20 mm, 40 mm, 60 mm or 85 mm); the sample output channel 12 converges in the middle of the microfluidic chip, and the angle formed is 30° to 120° (for example, 30°, 60°, 90° or 120°).

[0025] Preferably, the angle formed by the waste liquid output channel 11 and the sample output channel 12 is 15° to 60° (eg, 15°, 30°, 45° or 60°).

[0026] Preferably, the length of the middle microfluidic channel layer 2 is 65 to 265 mm (e.g., 65 mm, 100 mm, 200 mm or 265 mm), the width is 35 to 60 mm (e.g., 35 mm, 40 mm, 50 mm or 60 mm), and the thickness is 50 to 400 μm (e.g., 50 μm, 100 μm, 300 μm or 400 μm).

[0027] Preferably, the width of the rectangular window 13 is 10 to 54 mm (for example, 10 mm, 20 mm, 40 mm or 54 mm), and the length is 35 to 150 mm (for example, 35 mm, 50 mm, 100 mm or 150 mm). The rectangular window 13 is 0.5 to 10 mm (for example, 0.5 mm, 1 mm, 5 mm or 10 mm) away from the inner channel walls of the main flow channels 10 on both sides, 0 to 1 mm (for example, 0 mm, 0.3 mm, 0.5 mm or 1 mm) away from the confluence of the main flow channel 10, and 0 to 10 mm (for example, 0 mm, 3 mm, 5 mm or 10 mm) away from the branch outlet of the main flow channel 10.

[0028] In a second aspect, the present invention provides a system for capturing, identifying, counting and classifying cells, the system comprising the microfluidic chip described in the first aspect.

[0029] Preferably, the system further comprises magnetic nanoparticles linked to specific probes, fluorescent antibodies, a fluorescence microscope, an image processing device and a mechanical pump.

[0030] The system for cell capture, identification, counting, and classification described in this invention can enrich circulating tumor cells from large blood samples from cancer patients. A sample containing magnetic nanoparticles bound to target cells is injected into a microfluidic chip. Under the influence of the magnetic field and fluid resistance, the target cells migrate toward the sample output channel 12 and are collected at the sample outlet 6. Non-target cells migrate toward the waste fluid output channel 11 and are collected at the waste fluid outlet 7. The collected sample solution can be counted using a fluorescence microscope and used for downstream analysis.

[0031] In the present invention, the sample is derived from the blood or cell suspension of a cancer patient.

[0032] In the present invention, the magnetic nanoparticles are magnetic beads labeled with specific probes. The specific probes are antibodies or polypeptides of a specific protein, which can identify and capture cells that highly express the specific protein. In the present invention, different antibodies or polypeptides can be selected as probes for different proteins.

[0033] In the present invention, the sample is first pre-treated, a blood sample is obtained from a cancer patient, and nano-magnetic beads are added for incubation. The magnetic beads can specifically bind to the target cells. The sample is injected into the microfluidic chip. At the optimal flow rate, the sample is pushed into the sample inlet 4 by a mechanical pump and a syringe, and then enters the sample input flow channel 8, evenly shunting to the left and right sides. At the same time, a buffer solution (such as PBS solution) is pushed into the buffer inlet 5 by another mechanical pump and a syringe, and then enters the buffer input flow channel 9, evenly shunting to the left and right sides. After the sample and buffer solution flow into the main flow channel 10, they occupy the outside (sample layer) 15 and the inside (buffer layer) 16 of the flow channel respectively, presenting a laminar flow with clear boundaries. When the sample flows through the main flow channel 10, under the action of an external magnetic field, because the target cells are captured by the magnetic beads, the target cells subjected to the magnetic force will deviate from the magnet, move from the outside (sample layer) 15 of the main flow channel to the inside (buffer layer) 16, and then flow to the sample output flow channel under the drive of the buffer solution, and finally be collected at the sample outlet. Non-target cells not captured by the magnetic beads are not affected by the magnetic field and flow along with the sample solution to the waste liquid output channel 11, ultimately flowing out of the waste liquid outlet 7 and being discarded. The collected target cells are then stained with one or more fluorescently labeled antibodies and nuclear dyes. The cell suspension is then placed on a glass slide or well plate and counted using a matching fluorescence microscope and analysis software. Alternatively, the collected target cells can be directly used for subsequent genetic analysis.

[0034] By incorporating microfabrication and microfluidic chip technologies, this invention has developed a simple, easy-to-use, multilayer, embedded chip. This allows for the enrichment and sorting of circulating tumor cells in the complex blood environment while removing interference from non-target cells. This provides a new approach for liquid biopsy, enabling more efficient and rapid processing of large volumes of cancer patient blood and the preparation of highly pure and active circulating tumor cell suspensions for enumeration and subsequent downstream analysis.

[0035] In a third aspect, the present invention provides use of the microfluidic chip described in the first aspect or the system for cell capture, identification, counting and classification described in the second aspect in the preparation of a product for sorting and enriching circulating tumor cells.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The present invention achieves high-throughput, efficient sorting and enrichment, counting and downstream analysis of rare circulating tumor cells in blood. The present invention adopts dynamic magnetic sorting, which can sort circulating tumor cells from cancer patient blood samples faster than traditional magnetic adsorption. At the same time, the two parallel main flow channels 10 improve the processing throughput. Generally, it only takes 8 minutes from the injection of 2 mL sample into the chip to the completion of sorting. In addition, the present invention is simple to operate and causes little damage to the sample. Therefore, the enriched circulating tumor cells can be used for counting and have good cell activity, which can be used for subsequent downstream analysis.

[0038] (2) The present invention can remove the interference of complex non-target cells and impurities in the blood background. Traditional magnetic enrichment of circulating tumor cells mainly uses a magnetic frame to adsorb target cells to the tube wall or flow channel wall. Non-specific adsorption and physical deposition cause a large number of non-target cells and impurities to be retained, which causes great inconvenience to subsequent cell counting and downstream analysis. The present invention combines magnetic force and fluid resistance. Under the action of magnetic force, circulating tumor cells move from the outside of the fluid to the inside of the fluid, so that they are screened out from a large number of cells. Non-target cells also maintain their original flow trajectory under the fluid resistance and are taken out from the outside of the fluid, ultimately achieving the purpose of removing non-target cells and impurities, which provides feasibility for subsequent counting and downstream analysis;

[0039] (3) The chip and supporting system designed in the present invention are universal and highly automated. The nanomagnetic beads and fluorescent probes involved in the present invention can be adjusted according to the type of cancer and cell type. The core designed microfluidic chip can be used to sort and enrich circulating tumor cells under different sample conditions. It is highly flexible and low-cost. At the same time, the present invention has a high degree of automation. From sample injection to fluorescence imaging, to image processing and analysis, all are performed by pre-set sequencing, which greatly saves manpower and time. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the structure of the microfluidic chip of the present invention;

[0041] Figure 2 Schematic diagram of the cross-section of the middle microfluidic channel layer and the magnetic force action principle of the present invention;

[0042] Figure 3 Schematic diagram of the assembly of the microfluidic chip of the present invention;

[0043] Figure 4 This is a diagram showing the sorting effect of the microfluidic chip of the present invention;

[0044] Figure 5 This is a statistical diagram of the microfluidic chip sorting of the present invention.

[0045] In the figure, 1-top inlet and outlet layer; 2-middle microfluidic channel layer; 3-bottom substrate layer; 4-sample inlet; 5-buffer inlet; 6-sample outlet; 7-waste liquid outlet; 8-sample input channel; 9-buffer input channel; 10-main flow channel; 11-waste liquid output channel; 12-sample output channel; 13-empty window; 14-magnet; 15-outside (sample layer); 16-inside (buffer layer); 17-circulating tumor cells; 18-leukocytes; 19-red blood cells. DETAILED DESCRIPTION

[0046] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0047] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] Example 1

[0049] This embodiment provides a microfluidic chip for sorting and enriching circulating tumor cells in blood and a method for preparing the same.

[0050] The microfluidic chip of this embodiment comprises: a three-layer structure stacked together and sealed together, with a permanent magnet embedded therein, comprising, from top to bottom, a top inlet and outlet layer 1, a middle microfluidic channel layer 2, and a bottom base layer 3, with a rectangular window 13 opened in the middle of each layer;

[0051] The top inlet and outlet layer 1 is provided with a sample inlet 4, a buffer inlet 5, a sample outlet 6, and two waste liquid outlets 7; the top inlet and outlet layer 1 is connected to the middle microfluidic channel layer 2, and each inlet and outlet is connected to the corresponding inlet and outlet of each branch flow channel on the channel layer;

[0052] The middle microfluidic channel layer 2 is provided with two parallel main flow channels 10, two sample input flow channels 8, a buffer input flow channel 9, two waste liquid output flow channels 11 and two sample output flow channels 12;

[0053] Among them, the sample inlet 4 is connected to the sample input flow channel 8, and is split at the sample inlet 4, and is respectively merged into the two main flow channels 10 from both sides, and the flow channels are axially symmetrically distributed; the buffer inlet 5 is connected to the buffer input flow channel 9, and is split at the buffer inlet 5, and is respectively merged into the two main flow channels 10 from both sides, and the flow channels are axially symmetrically distributed; the waste liquid outlet 7 is connected to the waste liquid output flow channel 11, and flows from the main flow channels 10 on both sides into the waste liquid output flow channels 11 and the waste liquid outlet 7 on both sides respectively; the sample outlet 6 is connected to the sample output flow channel 12, and flows from the main flow channels 10 on both sides into the two sample output flow channels 12 in the middle, and the two sample output flow channels 12 converge at the sample outlet 6; wherein, the sample input flow channel 8 and the waste liquid output flow channel 11 are located on the outside of the overall flow channel, the buffer input flow channel 9 and the sample output flow channel 12 are located on the inside of the overall flow channel, and the entire structure is axially symmetrically distributed;

[0054] The bottom base layer 3 is a smooth, flat bottom plate; the rectangular window 13 is a hollow part opened in the middle of the main flow channel 10 after the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom base layer 3 are assembled, and the rectangular window 13 has a magnet 14 embedded in it.

[0055] The thicknesses of the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom substrate layer 3 are 3500 μm, 100 μm, and 200 μm, respectively. The top inlet and outlet layer 1 and the middle microfluidic channel layer 2 are located on the upper and lower surfaces of the same piece of material, respectively. The inlet and outlet of the top inlet and outlet layer 1 are connected to the flow channel inlet and outlet of the microfluidic channel layer 2. During the manufacturing process, they are formed in one step, eliminating the need for separate manufacturing and packaging.

[0056] The top needs to be connected to the solution input and output tubes for connecting syringes.

[0057] The bottom base layer 3 is a smooth surface for easy cutting and is bonded to the middle microfluidic channel layer 2 .

[0058] Since the chip needs to have embedded magnets, insulating polymers with good toughness are particularly preferred.

[0059] The top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom substrate layer 3 are all made of PDMS (polydimethylsiloxane) and are sealed and connected by bonding. In the middle microfluidic channel layer 2, the narrowest point of the channel wall is 200 μm, and the channel depth is 100 μm.

[0060] In this embodiment, the main flow channels 10 on both sides of the middle microfluidic channel layer 2 have a width of 1500 μm and a length of 73 mm. The inner flow channel walls of the main flow channels 10 on both sides are 44 mm apart and are parallel and symmetrically distributed.

[0061] In this embodiment, the sample input channel 8 has a channel width of 500 μm, a height of 22.5 mm, and a width of 47 mm. The inner radius of the arc-shaped channel of the sample input channel 8 is 6 mm. The buffer input channel 9 has a channel width of 800 μm, a height of 18.3 mm, and a width of 45.6 mm. The inner radius of the arc-shaped channel of the buffer input channel 9 is 10.1 mm.

[0062] In this embodiment, the shortest distance between the sample input flow channel 8 and the buffer input flow channel 9 at the confluence of the main flow channel 10 is 200 μm.

[0063] In this embodiment, the waste liquid output channel 11 has a channel width of 700 μm and a height of 40.9 mm.

[0064] In this embodiment, the sample output flow channel 12 has a flow channel width of 800 μm and a height of 40.9 mm. The two sample output flow channels 12 merge in the middle of the microfluidic chip, and the angle formed therebetween is 60°.

[0065] In this embodiment, the angle formed by the waste liquid output flow channel 11 and the sample output flow channel 12 is 30°.

[0066] 2. Rectangular window and inner magnet

[0067] In this embodiment, the cross-section of the middle microfluidic channel layer and the schematic diagram of the magnetic force action principle are shown in FIG. Figure 2 As shown, the rectangular window 13 and the embedded magnet 14 are key components for sorting cells. In order to provide a uniform and stable magnetic field, the magnet used is a regular rectangular permanent magnet, and the size of the magnet is consistent with the rectangular window.

[0068] Each layer of the chip contains a rectangular window 13, 33mm wide and 62mm long, extending through the entire chip. A magnet 14 is fully embedded within the window 13. The left and right sides of the window 13 (the magnet's polar surfaces) are 5.5mm away from the inner walls 16 of the main flow channel 10, 1mm away from the confluence of the main flow channel, and 10mm away from the branching port of the main flow channel.

[0069] 3. Sealing method

[0070] like Figure 3 As shown, the materials of the top inlet and outlet layer 1, the middle microfluidic channel layer 2 and the bottom substrate layer 3 are all PDMS (polydimethylsiloxane) materials, and oxygen plasma bonding method is used to achieve a good sealing connection between the PDMS.

[0071] 4. Chip supporting system

[0072] This embodiment also provides a system for identifying and counting the sorted and enriched cells, which includes, in addition to the microfluidic chip of the present invention, a fluorescent probe, a fluorescent microscope, image processing software, a mechanical pump, and a syringe.

[0073] Fluorescent probes are used to identify cells. In this example, pan-CK keratin antibodies labeled with green fluorescence are used to identify circulating tumor cells; CD45 transmembrane protein antibodies labeled with red fluorescence are used to identify leukocytes; and Hoechst dye is used to stain cell nuclei.

[0074] Fluorescence microscopy is used to detect whether there are circulating tumor cells in the sorted and enriched solution, and whether the cells bind to each fluorescent probe.

[0075] Image processing software is used to analyze images captured by fluorescence microscopy and determine the corresponding circulating tumor cell counts. The software can identify and count qualifying circulating tumor cells based on nuclear size, binding of different fluorescent probes, and brightfield images.

[0076] Mechanical pumps and syringes are used to inject samples and related reagents.

[0077] 5. Fabrication Method of Microfluidic Chip

[0078] The microfluidic chip is manufactured using the following manufacturing method to help those skilled in the art understand the manufacturing method of the present invention, but is not intended to limit the material, size and manufacturing method of the microfluidic chip of the present invention.

[0079] Top inlet and outlet layer 1 and middle microfluidic channel layer 2: Using an N-type 4-inch silicon wafer, the desired pattern is transferred to the wafer using photolithography. The resulting wafer is then processed using inductively coupled plasma etching, a technique commonly used in semiconductors. A convex chip with the inverse of the desired pattern is etched, serving as a mold. A prepared PDMS (a mixture of the basic components of Sylgard 184 and a curing agent in a 10:1 weight ratio) colloid is poured onto the finished chip mold and placed in a vacuum drying oven for degassing and curing, resulting in an internally uniform, resilient solid structure. Once fully solidified, the mold is removed, and the excess portion not containing the flow channel is removed. A punch is then used to drill through holes at the designated inlet and outlet locations, resulting in the top inlet and outlet layer 1 and middle microfluidic channel layer 2.

[0080] Bottom substrate layer: Pour the prepared PDMS colloid into a clean, flat container and place it in a vacuum drying oven for degassing and curing, resulting in a uniform, elastic solid structure. Once completely solidified, remove the mold and cut out a piece of PDMS solid the same size as the top inlet and outlet layer 1 and the middle microfluidic channel layer 2 to form the bottom substrate layer 3.

[0081] Chip assembly and bonding: After ultrasonic cleaning of the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom substrate layer 3, they are treated using an oxygen plasma etcher. The flat side of the PDMS substrate is selected, while the side with flow channels is selected for the top inlet and outlet layer 1 and the middle microfluidic channel layer 2. The treated surfaces are bonded together and bonded through a high-temperature bake to form the final chip.

[0082] The chip system: Based on the aforementioned microfluidic chip, a needle is inserted into the inlet and outlet, and plastic tubing is used to connect the syringe and sample collection container. A mechanical pump propels the syringe at a set flow rate, pushing the solution into the microfluidic chip. The sorted and enriched samples are placed under a fluorescence microscope and analyzed and processed using image processing software or used directly for downstream genetic analysis, completing the entire system.

[0083] Before use, the chip can be disinfected by spraying the surface with 75% alcohol and injecting it into the chip using a syringe. The chip is then washed with a buffer solution (PBS). To prevent nonspecific adsorption of cells to the channel walls, a PBS solution containing 5% BSA is filled into the microfluidic channels using a mechanical pump at a low flow rate for 1 hour. After these treatments, the chip can be used for subsequent experiments.

[0084] Example 2

[0085] This embodiment provides a microfluidic chip for sorting and enriching circulating tumor cells in blood and a method for preparing the same.

[0086] The microfluidic chip of this embodiment comprises: a three-layer structure stacked together and sealed together, with a permanent magnet embedded therein, comprising, from top to bottom, a top inlet and outlet layer 1, a middle microfluidic channel layer 2, and a bottom base layer 3, with a rectangular window 13 opened in the middle of each layer;

[0087] The top inlet and outlet layer 1 is provided with a sample inlet 4, a buffer inlet 5, a sample outlet 6, and two waste liquid outlets 7; the top inlet and outlet layer 1 is connected to the middle microfluidic channel layer 2, and each inlet and outlet is connected to the corresponding inlet and outlet of each branch flow channel on the channel layer;

[0088] The middle microfluidic channel layer 2 is provided with two parallel main flow channels 10, two sample input flow channels 8, a buffer input flow channel 9, two waste liquid output flow channels 11 and two sample output flow channels 12;

[0089] Among them, the sample inlet 4 is connected to the sample input flow channel 8, and is split at the sample inlet 4, and is respectively merged into the two main flow channels 10 from both sides, and the flow channels are axially symmetrically distributed; the buffer inlet 5 is connected to the buffer input flow channel 9, and is split at the buffer inlet 5, and is respectively merged into the two main flow channels 10 from both sides, and the flow channels are axially symmetrically distributed; the waste liquid outlet 7 is connected to the waste liquid output flow channel 11, and flows from the main flow channels 10 on both sides into the waste liquid output flow channels 11 and the waste liquid outlet 7 on both sides respectively; the sample outlet 6 is connected to the sample output flow channel 12, and flows from the main flow channels 10 on both sides into the two sample output flow channels 12 in the middle, and the two sample output flow channels 12 converge at the sample outlet 6; wherein, the sample input flow channel 8 and the waste liquid output flow channel 11 are located on the outside of the overall flow channel, the buffer input flow channel 9 and the sample output flow channel 12 are located on the inside of the overall flow channel, and the entire structure is axially symmetrically distributed;

[0090] The bottom base layer 3 is a smooth, flat bottom plate; the rectangular window 13 is a hollow part opened in the middle of the main flow channel 10 after the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom base layer 3 are assembled, and the rectangular window 13 has a magnet 14 embedded in it.

[0091] The thicknesses of the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom substrate layer 3 are 4500 μm, 50 μm, and 100 μm, respectively. The top inlet and outlet layer 1 and the middle microfluidic channel layer 2 are located on the upper and lower surfaces of the same piece of material, respectively. The inlet and outlet of the top inlet and outlet layer 1 are connected to the flow channel inlet and outlet of the microfluidic channel layer 2. They are formed in one step during fabrication, eliminating the need for separate manufacturing and subsequent packaging.

[0092] The top needs to be connected to the solution input and output tubes for connecting syringes.

[0093] The bottom base layer 3 is a smooth surface for easy cutting and is bonded to the middle microfluidic channel layer 2 .

[0094] Since the chip needs to have embedded magnets, insulating polymers with good toughness are particularly preferred.

[0095] The top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom substrate layer 3 are all made of PDMS (polydimethylsiloxane) and are sealed and connected by bonding. In the middle microfluidic channel layer 2, the narrowest point of the channel wall is 100 μm, and the channel depth is 50 μm.

[0096] In this embodiment, the main flow channels 10 on both sides of the middle microfluidic channel layer 2 have a width of 750 μm and a length of 35 mm. The inner channel walls of the main flow channels 10 on both sides are 33.5 mm apart and are parallel and symmetrically distributed.

[0097] In this embodiment, the sample input channel 8 has a channel width of 250 μm, a height of 10 mm, and a width of 35 mm, and the inner radius of the arc-shaped channel of the sample input channel 8 is 3 mm; the buffer input channel 9 has a channel width of 400 μm, a height of 5 mm, and a width of 34.3 mm, and the inner radius of the arc-shaped channel of the buffer input channel 9 is 5 mm.

[0098] In this embodiment, the shortest distance between the sample input flow channel 8 and the buffer input flow channel 9 at the confluence of the main flow channel 10 is 100 μm.

[0099] In this embodiment, the waste liquid output channel 11 has a channel width of 350 μm and a height of 64 mm.

[0100] In this embodiment, the sample output flow channel 12 has a flow channel width of 400 μm and a height of 64 mm. The sample output flow channel 12 merges in the middle of the microfluidic chip, and the angle formed is 30°.

[0101] In this embodiment, the angle formed by the waste liquid output flow channel 11 and the sample output flow channel 12 is 15°.

[0102] 2. Rectangular window and inner magnet

[0103] In this embodiment, the cross-section of the middle microfluidic channel layer and the schematic diagram of the magnetic force action principle are shown in FIG. Figure 2 As shown, the rectangular window 13 and the embedded magnet 14 are key components for sorting cells. In order to provide a uniform and stable magnetic field, the magnet used is a regular rectangular permanent magnet, and the size of the magnet is consistent with the rectangular window.

[0104] Each layer of the chip contains a rectangular window 13, 13.5mm wide and 29.5mm long, extending through the entire chip. A magnet 14 is fully embedded within the window 13. The left and right sides of the window 13 (the magnet's polar surfaces) are 10mm away from the inner walls 16 of the main flow channel 10, 0.5mm from the confluence of the main flow channel, and 5mm from the branching port.

[0105] 3. Sealing method

[0106] like Figure 3 As shown, the materials of the top inlet and outlet layer 1, the middle microfluidic channel layer 2 and the bottom substrate layer 3 are all PDMS (polydimethylsiloxane) materials, and oxygen plasma bonding method is used to achieve a good sealing connection between the PDMS.

[0107] 4. Chip supporting system

[0108] This embodiment also provides a system for identifying and counting the sorted and enriched cells, which includes, in addition to the microfluidic chip of the present invention, a fluorescent probe, a fluorescent microscope, image processing software, a mechanical pump, and a syringe.

[0109] Fluorescent probes are used to identify cells. In this example, pan-CK keratin antibodies labeled with green fluorescence are used to identify circulating tumor cells; CD45 transmembrane protein antibodies labeled with red fluorescence are used to identify leukocytes; and Hoechst dye is used to stain cell nuclei.

[0110] Fluorescence microscopy is used to detect whether there are circulating tumor cells in the sorted and enriched solution, and whether the cells bind to each fluorescent probe.

[0111] Image processing software is used to analyze images captured by fluorescence microscopy and determine the corresponding circulating tumor cell counts. The software can identify and count qualifying circulating tumor cells based on nuclear size, binding of different fluorescent probes, and brightfield images.

[0112] Mechanical pumps and syringes are used to inject samples and related reagents.

[0113] 5. Fabrication Method of Microfluidic Chip

[0114] The microfluidic chip is manufactured using the following manufacturing method to help those skilled in the art understand the manufacturing method of the present invention, but is not intended to limit the material, size and manufacturing method of the microfluidic chip of the present invention.

[0115] Top inlet and outlet layer 1 and middle microfluidic channel layer 2: Use N-type 4-inch silicon wafers, use photolithography technology to transfer the required pattern to the 4-inch silicon wafer, and use inductively coupled plasma etching commonly used in semiconductors to process the silicon wafer after photolithography. The convex chip etched out with the opposite pattern to the required pattern is used as a mold. The configured PDMS colloid is poured onto the processed chip mold and placed in a vacuum drying oven for degassing and curing to make it an internally uniform and elastic solid structure. When it is completely solidified, it is demolded, the excess part that does not contain the flow channel is cut off, and a punch is used to punch through according to the set inlet and outlet positions to obtain the top inlet and outlet layer 1 and the middle microfluidic channel layer 2.

[0116] Bottom substrate layer: Pour the prepared PDMS colloid into a clean, flat container and place it in a vacuum drying oven for degassing and curing, resulting in a uniform, elastic solid structure. Once completely solidified, remove the mold and cut out a piece of PDMS solid the same size as the top inlet and outlet layer 1 and the middle microfluidic channel layer 2 to form the bottom substrate layer 3.

[0117] Chip assembly and bonding: After ultrasonic cleaning of the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom substrate layer 3, they are treated using an oxygen plasma etcher. The flat side of the PDMS substrate is selected, while the side with flow channels is selected for the top inlet and outlet layer 1 and the middle microfluidic channel layer 2. The treated surfaces are bonded together and bonded through a high-temperature bake to form the final chip.

[0118] The chip system: Based on the aforementioned microfluidic chip, a needle is inserted into the inlet and outlet, and plastic tubing is used to connect the syringe and sample collection container. A mechanical pump propels the syringe at a set flow rate, pushing the solution into the microfluidic chip. The sorted and enriched samples are placed under a fluorescence microscope and analyzed and processed using image processing software or used directly for downstream genetic analysis, completing the entire system.

[0119] Before use, the chip can be disinfected by spraying the surface with 75% alcohol and injecting it into the chip using a syringe. The chip is then washed with a buffer solution (PBS). To prevent nonspecific adsorption of cells to the channel walls, a PBS solution containing 5% BSA is filled into the microfluidic channels using a mechanical pump at a low flow rate for 1 hour. After these treatments, the chip can be used for subsequent experiments.

[0120] Example 3

[0121] This embodiment provides a microfluidic chip for sorting and enriching circulating tumor cells in blood and a method for preparing the same.

[0122] The microfluidic chip of this embodiment comprises: a three-layer structure stacked together and sealed together, with a permanent magnet embedded therein, comprising, from top to bottom, a top inlet and outlet layer 1, a middle microfluidic channel layer 2, and a bottom base layer 3, with a rectangular window 13 opened in the middle of each layer;

[0123] The top inlet and outlet layer 1 is provided with a sample inlet 4, a buffer inlet 5, a sample outlet 6, and two waste liquid outlets 7; the top inlet and outlet layer 1 is connected to the middle microfluidic channel layer 2, and each inlet and outlet is connected to the corresponding inlet and outlet of each branch flow channel on the channel layer;

[0124] The middle microfluidic channel layer 2 is provided with two parallel main flow channels 10, two sample input flow channels 8, a buffer input flow channel 9, two waste liquid output flow channels 11 and two sample output flow channels 12;

[0125] Among them, the sample inlet 4 is connected to the sample input flow channel 8, and is split at the sample inlet 4, and is respectively merged into the two main flow channels 10 from both sides, and the flow channels are axially symmetrically distributed; the buffer inlet 5 is connected to the buffer input flow channel 9, and is split at the buffer inlet 5, and is respectively merged into the two main flow channels 10 from both sides, and the flow channels are axially symmetrically distributed; the waste liquid outlet 7 is connected to the waste liquid output flow channel 11, and flows from the main flow channels 10 on both sides into the waste liquid output flow channels 11 and the waste liquid outlet 7 on both sides respectively; the sample outlet 6 is connected to the sample output flow channel 12, and flows from the main flow channels 10 on both sides into the two sample output flow channels 12 in the middle, and the two sample output flow channels 12 converge at the sample outlet 6; wherein, the sample input flow channel 8 and the waste liquid output flow channel 11 are located on the outside of the overall flow channel, the buffer input flow channel 9 and the sample output flow channel 12 are located on the inside of the overall flow channel, and the entire structure is axially symmetrically distributed;

[0126] The bottom base layer 3 is a smooth, flat bottom plate; the rectangular window 13 is a hollow part opened in the middle of the main flow channel 10 after the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom base layer 3 are assembled, and the rectangular window 13 has a magnet 14 embedded in it.

[0127] The thicknesses of the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom substrate layer 3 are 900 μm, 400 μm, and 2000 μm, respectively. The top inlet and outlet layer 1 and the middle microfluidic channel layer 2 are located on the upper and lower surfaces of the same piece of material, respectively. The inlet and outlet of the top inlet and outlet layer 1 are connected to the flow channel inlet and outlet of the microfluidic channel layer 2. During the manufacturing process, they are formed in one step, eliminating the need for separate manufacturing and packaging.

[0128] The top needs to be connected to the solution input and output tubes for connecting syringes.

[0129] The bottom base layer 3 is a smooth surface for easy cutting and is bonded to the middle microfluidic channel layer 2 .

[0130] Since the chip needs to have embedded magnets, insulating polymers with good toughness are particularly preferred.

[0131] The top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom substrate layer 3 are all made of PDMS (polydimethylsiloxane) and are sealed and connected by bonding. In the middle microfluidic channel layer 2, the narrowest point of the channel wall is 400 μm, and the channel depth is 400 μm.

[0132] In this embodiment, the main flow channels 10 on both sides of the middle microfluidic channel layer 2 have a width of 3000 μm and a length of 150 mm. The inner flow channel walls of the main flow channels 10 on both sides are 55 mm apart and are parallel and symmetrically distributed.

[0133] In this embodiment, the sample input channel 8 has a channel width of 1000 μm, a height of 30 mm, and a width of 60 mm, and the inner radius of the arc-shaped channel of the sample input channel 8 is 10 mm; the buffer input channel 9 has a channel width of 1600 μm, a height of 25 mm, and a width of 57.2 mm, and the inner radius of the arc-shaped channel of the buffer input channel 9 is 20 mm.

[0134] In this embodiment, the shortest distance between the sample input flow channel 8 and the buffer input flow channel 9 at the confluence of the main flow channel 10 is 400 μm.

[0135] In this embodiment, the waste liquid output channel 11 has a channel width of 1400 μm and a height of 20 mm.

[0136] In this embodiment, the sample output flow channel 12 has a flow channel width of 1600 μm and a height of 20 mm. The sample output flow channel 12 merges in the middle of the microfluidic chip, and the angle formed is 120°.

[0137] In this embodiment, the angle formed by the waste liquid output flow channel 11 and the sample output flow channel 12 is 60°.

[0138] 2. Rectangular window and inner magnet

[0139] In this embodiment, the cross-section of the middle microfluidic channel layer and the schematic diagram of the magnetic force action principle are shown in FIG. Figure 2 As shown, the rectangular window 13 and the embedded magnet 14 are key components for sorting cells. In order to provide a uniform and stable magnetic field, the magnet used is a regular rectangular permanent magnet, and the size of the magnet is consistent with the rectangular window.

[0140] Each layer of the chip contains a rectangular window 13, 35mm wide and 139mm long, extending through the entire chip. A magnet 14 is fully embedded within the window 13. The left and right sides of the window 13 (the magnet's polar surfaces) are 10mm away from the inner walls 16 of the main flow channel 10, 1mm away from the confluence of the main flow channel, and 10mm away from the branching port of the main flow channel.

[0141] 3. Sealing method

[0142] like Figure 3 As shown, the materials of the top inlet and outlet layer 1, the middle microfluidic channel layer 2 and the bottom substrate layer 3 are all PDMS (polydimethylsiloxane) materials, and oxygen plasma bonding method is used to achieve a good sealing connection between the PDMS.

[0143] 4. Chip supporting system

[0144] This embodiment also provides a system for identifying and counting the sorted and enriched cells, which includes, in addition to the microfluidic chip of the present invention, a fluorescent probe, a fluorescent microscope, image processing software, a mechanical pump, and a syringe.

[0145] Fluorescent probes are used to identify cells. In this example, pan-CK keratin antibodies labeled with green fluorescence are used to identify circulating tumor cells; CD45 transmembrane protein antibodies labeled with red fluorescence are used to identify leukocytes; and Hoechst dye is used to stain cell nuclei.

[0146] Fluorescence microscopy is used to detect whether there are circulating tumor cells in the sorted and enriched solution, and whether the cells bind to each fluorescent probe.

[0147] Image processing software is used to analyze images captured by fluorescence microscopy and determine the corresponding circulating tumor cell counts. The software can identify and count qualifying circulating tumor cells based on nuclear size, binding of different fluorescent probes, and brightfield images.

[0148] Mechanical pumps and syringes are used to inject samples and related reagents.

[0149] 5. Fabrication Method of Microfluidic Chip

[0150] The microfluidic chip is manufactured using the following manufacturing method to help those skilled in the art understand the manufacturing method of the present invention, but is not intended to limit the material, size and manufacturing method of the microfluidic chip of the present invention.

[0151] Top inlet and outlet layer 1 and middle microfluidic channel layer 2: Use N-type 4-inch silicon wafers, use photolithography technology to transfer the required pattern to the 4-inch silicon wafer, and use inductively coupled plasma etching commonly used in semiconductors to process the silicon wafer after photolithography. The convex chip etched out with the opposite pattern to the required pattern is used as a mold. The configured PDMS colloid is poured onto the processed chip mold and placed in a vacuum drying oven for degassing and curing to make it an internally uniform and elastic solid structure. When it is completely solidified, it is demolded, the excess part that does not contain the flow channel is cut off, and a punch is used to punch through according to the set inlet and outlet positions to obtain the top inlet and outlet layer 1 and the middle microfluidic channel layer 2.

[0152] Bottom substrate layer: Pour the prepared PDMS colloid into a clean, flat container and place it in a vacuum drying oven for degassing and curing, resulting in a uniform, elastic solid structure. Once completely solidified, remove the mold and cut out a piece of PDMS solid the same size as the top inlet and outlet layer 1 and the middle microfluidic channel layer 2 to form the bottom substrate layer 3.

[0153] Chip assembly and bonding: After ultrasonic cleaning of the top inlet and outlet layer 1, the middle microfluidic channel layer 2, and the bottom substrate layer 3, they are treated using an oxygen plasma etcher. The flat side of the PDMS substrate is selected, while the side with flow channels is selected for the top inlet and outlet layer 1 and the middle microfluidic channel layer 2. The treated surfaces are bonded together and bonded through a high-temperature bake to form the final chip.

[0154] The chip system: Based on the aforementioned microfluidic chip, a needle is inserted into the inlet and outlet, and plastic tubing is used to connect the syringe and sample collection container. A mechanical pump propels the syringe at a set flow rate, pushing the solution into the microfluidic chip. The sorted and enriched samples are placed under a fluorescence microscope and analyzed and processed using image processing software or used directly for downstream genetic analysis, completing the entire system.

[0155] Before use, the chip can be disinfected by spraying the surface with 75% alcohol and injecting it into the chip using a syringe. The chip is then washed with a buffer solution (PBS). To prevent nonspecific adsorption of cells to the channel walls, a PBS solution containing 5% BSA is filled into the microfluidic channels using a mechanical pump at a low flow rate for 1 hour. After these treatments, the chip can be used for subsequent experiments.

[0156] Test Example 1

[0157] The microfluidic chip prepared in Examples 1-3 was used to perform sorting, enrichment, counting and downstream analysis of circulating tumor cells in the blood.

[0158] Artificial blood samples were prepared by mixing MCF-7 cancer cells and white blood cells into 2 mL of PBS. The blood sample was diluted 5-fold with PBS containing 5% BSA, and nanomagnetic beads linked to EpCAM antibodies were added, mixed thoroughly, and incubated for 1 hour. The incubated sample was then placed on a horizontal magnetic stand. The cell-bead mixture was deposited at the bottom by magnetic force and gravity. The 8 mL of waste liquid above was discarded, and 2 mL of pretreated sample was used. The pretreated sample was then introduced into the microfluidic chip through sample inlet 4, while PBS solution was simultaneously introduced through buffer inlet 5. Within the microfluidic chip, under the influence of the magnetic field, circulating tumor cells captured by the magnetic beads moved from the outer side (sample layer) 15 of the main flow channel to the inner side (PBS solution layer) 16. There, driven by the PBS solution, they flowed into the sample outlet channel 12 and were finally collected at sample outlet 6. The sorted and enriched circulating tumor cells were stained with green fluorescent pan-CK keratin antibodies, red fluorescent CD45 transmembrane protein antibodies, and blue Hoechst fluorescent dye. Among them, pan-CK keratin is a marker of cancer cells MCF-7, CD45 is a marker of white blood cells, and Hoechst fluorescent dye is used to stain the cell nucleus. The stained cell suspension is dropped into the well plate, and the image is taken using a fluorescence microscope, and the image processing software is used to analyze and count. Example 1 Sorting results are shown in FIG. Figure 4 As shown, cells that simultaneously emit blue and green fluorescence signals are marked as circulating tumor cells; cells that simultaneously emit blue and red fluorescence signals are marked as white blood cells. After the artificial blood sample is processed by the microfluidic chip, the cell suspension collected at the sample outlet 6 is mostly MCF-7 cells with only a small amount of white blood cells, while the waste liquid outlet 7 is the opposite. The statistical results of the sorting in Example 1 are shown in Figure 1. Figure 5 As shown, at the optimal flow rate (sample input channel flow rate: 250 μL / min, buffer input channel flow rate: 450 μL / min), the leukocyte (non-target cell) removal rate of the microfluidic chip can reach 99.55%; the MCF-7 sorting efficiency can reach 98.50%, and the purity of the collected MCF-7 cell suspension is 99.68%. In addition, the circulating tumor cells sorted and enriched by the microfluidic chip can be used for subsequent downstream analysis: 10×Genomics single-cell transcriptome sequencing, single-cell DNA methylation group sequencing, etc. The statistical results of Examples 2 and 3 show that at the optimal flow rate (sample input flow channel flow rate: 250 μL / min, buffer input flow channel flow rate: 450 μL / min), the leukocyte (non-target cell) removal rate of the microfluidic chip can reach 98.51% and 94.60%, respectively; the MCF-7 sorting efficiency can reach 92.36% and 96.91%, respectively, and the purity of the collected MCF-7 cell suspensions is 91.79% and 90.66%, respectively.

[0159] In summary, the present invention introduces micromachining technology and microfluidic chip technology to develop a simple and easy-to-use multi-layer, embedded structure microfluidic chip. It can enrich and sort circulating tumor cells in a complex blood environment and remove interference from non-target cells. It provides a new method for the field of liquid biopsy, which can more efficiently and quickly process large amounts of cancer patient blood and collect circulating tumor cells for counting and subsequent downstream analysis.

[0160] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A microfluidic chip, characterized in that: The microfluidic chip comprises: a three-layer structure stacked together and sealed with each other, and embedded with a permanent magnet, wherein from top to bottom, the three layers are respectively a top inlet and outlet layer (1), a middle microfluidic channel layer (2), and a bottom base layer (3), and a rectangular window (13) is opened in the middle of each layer; The top inlet and outlet layer (1) is provided with a sample inlet (4), a buffer inlet (5), a sample outlet (6), and two waste liquid outlets (7); the top inlet and outlet layer (1) is connected to the middle microfluidic channel layer (2), and each inlet and outlet is connected to the corresponding inlet and outlet of each branch flow channel on the channel layer; The middle microfluidic channel layer (2) is provided with two parallel main flow channels (10), two sample input flow channels (8), a buffer input flow channel (9), two waste liquid output flow channels (11) and two sample output flow channels (12); The sample inlet (4) is connected to the sample input flow channel (8), and the flow is divided at the sample inlet (4) and respectively merged into two main flow channels (10) from both sides, and the flow channels are symmetrically distributed; the buffer inlet (5) is connected to the buffer input flow channel (9), and the flow is divided at the buffer inlet (5) and respectively merged into two main flow channels (10) from both sides, and the flow channels are symmetrically distributed; the waste liquid outlet (7) is connected to the waste liquid output flow channel (11), and the waste liquid flows from the main flow channels (10) on both sides into the waste liquid output flow channels on both sides. The sample outlet (6) is connected to the sample output flow channel (12), and the liquid flows from the main flow channels (10) on both sides into the two sample output flow channels (12) in the middle, and the two sample output flow channels (12) merge at the sample outlet (6); wherein, the sample input flow channel (8) and the waste liquid output flow channel (11) are located outside the overall flow channel, and the buffer input flow channel (9) and the sample output flow channel (12) are located inside the overall flow channel, and the entire structure is axially symmetrically distributed; The bottom base layer (3) is a smooth, flat bottom plate; the rectangular window (13) is a hollow portion opened in the middle of the main flow channel (10) after the top inlet and outlet layer (1), the middle microfluidic channel layer (2), and the bottom base layer (3) are assembled, and the rectangular window (13) is embedded with a magnet (14).

2. The microfluidic chip according to claim 1, characterized in that The diameters of the five inlets and outlets of the top inlet and outlet layer (1) are 1000 to 4000 μm; the thickness of the top inlet and outlet layer (1) is 900 to 4500 μm.

3. The microfluidic chip according to claim 1 or 2, characterized in that: The flow channel depth of the middle microfluidic channel layer (2) is 50 to 400 μm; Preferably, the main flow channels (10) on both sides of the middle microfluidic channel layer (2) have a width of 750 to 3000 μm and a length of 35 to 150 mm, and the inner flow channel walls of the main flow channels (10) on both sides are 30 to 55 mm apart and are distributed in parallel and symmetrical manner.

4. The microfluidic chip according to any one of claims 1 to 3, characterized in that The sample input flow channel (8) has a flow channel width of 250 to 1000 μm, a height of 10 to 30 mm, and a width of 35 to 60 mm, and the inner radius of the arc-shaped flow channel of the sample input flow channel (8) is 3 to 10 mm; the buffer input flow channel (9) has a flow channel width of 400 to 1600 μm, a height of 5 to 25 mm, and a width of 34.3 to 57.2 mm, and the inner radius of the arc-shaped flow channel of the buffer input flow channel (9) is 5 to 20 mm.

5. The microfluidic chip according to any one of claims 1 to 4, characterized in that: The shortest distance between the sample input flow channel (8) and the buffer input flow channel (9) at the confluence entrance of the main flow channel (10) is 100 to 400 μm.

6. The microfluidic chip according to any one of claims 1 to 5, characterized in that: The waste liquid output channel (11) has a channel width of 350 to 1400 μm and a height of 20 to 85 mm.

7. The microfluidic chip according to any one of claims 1 to 6, characterized in that: The sample output flow channel (12) has a flow channel width of 400 to 1600 μm and a height of 20 to 85 mm; the sample output flow channel (12) converges in the middle of the microfluidic chip, and the angle formed is 30° to 120°; Preferably, the angle formed by the waste liquid output flow channel (11) and the sample output flow channel (12) is 15° to 60°.

8. The microfluidic chip according to any one of claims 1 to 7, characterized in that: The middle microfluidic channel layer (2) has a length of 65 to 265 mm, a width of 35 to 60 mm, and a thickness of 50 to 400 μm; Preferably, the rectangular window (13) has a width of 10 to 54 mm and a length of 35 to 150 mm, and the rectangular window (13) is 0.5 to 10 mm away from the inner flow channel walls of the main flow channels (10) on both sides, 0 to 1 mm away from the confluence port of the main flow channel (10), and 0 to 10 mm away from the diversion port of the main flow channel (10).

9. A system for capturing, identifying, counting and sorting cells, characterized in that: The system comprises the microfluidic chip according to any one of claims 1 to 8; Preferably, the system further comprises magnetic nanoparticles linked to specific probes, fluorescent antibodies, a fluorescence microscope, an image processing device and a mechanical pump.

10. Use of the microfluidic chip according to any one of claims 1 to 8 or the system for cell capture, identification, counting and classification according to claim 9 in the preparation of a product for sorting and enriching circulating tumor cells.

Images