Tumor cell separation and capture microfluidic chip and use method

Abstract

The present invention belongs to the field of biophysics technology, and in particular relates to a tumor cell separation and capture microfluidic chip and a method of use, comprising a first microfluidic chip, wherein a first microchannel is provided in the first microfluidic chip, wherein a first microcolumn array is provided in the first microchannel; a second microfluidic chip, wherein a second microchannel is provided in the second microchannel, wherein a second microcolumn array is provided in the second microchannel; and a third microfluidic chip, wherein a third microchannel is provided in the third microfluidic chip, wherein a third microcolumn array is provided in the third microchannel, wherein the outlet of the third microcolumn array is connected to the inlet of a curved channel, and the curved channel is fixed in the third microchannel. When the present invention is used, the first microfluidic chip uses affinity to capture or remove white blood cells, and the second and third microfluidic chips use physical capture, thereby achieving dual capture of affinity and physical properties; different tumor cells and invasion distances are separated by setting up three layers of chips.

Description

Technical Field

[0001] The present invention belongs to the field of biophysics technology, and in particular relates to a microfluidic chip for separating and capturing tumor cells and a method for using the chip. Background Art

[0002] Circulating tumor cells (CTCs) are tumor cells that shed from primary or regenerating tumors and enter the bloodstream and lymphatic systems. These tumor cells then migrate to other locations with suitable growth environments, multiplying and developing into new tumors. The detection of circulating tumor cells in the bloodstream indicates cancer and possible metastasis. The number of CTCs is closely linked to the severity of the cancer. A high number indicates a serious condition, while a low number indicates milder disease or treatment effectiveness.

[0003] The microchannels of microfluidic chips are comparable in size to cells, making them ideal for cell separation and capture studies. Existing methods for capturing and separating CTCs using microfluidic chips can be broadly divided into two categories:

[0004] One type of method uses the affinity reaction principle to capture and separate cells. This method relies on modifying specific antibodies that can bind to cell surface antigens on the microchannels or microstructures inside the microfluidic chip, thereby improving capture efficiency and separation purity. However, the structure of this microfluidic chip is usually very complex, the modification time is long, the antibodies used are expensive, and the expression of epithelial cell adhesion molecules varies in different tumor cells. Moreover, when tumor cells undergo epithelial-mesenchymal transition (EMT), the expression of epithelial cell adhesion molecules in tumor cells decreases. Therefore, relying on this method to capture CTCs may lose some tumor cells that do not express or lowly express epithelial cell adhesion molecules.

[0005] Another type of capture method is based on physical properties, such as isolation by size of epithelial tumor cells (ISET). This method uses the fact that CTCs are larger than blood cells and less susceptible to deformation to filter through blood cells. This method is simple to operate, requires no complex structure, no modification, and does not rely on any surface markers. However, because CTCs and white blood cells overlap in size, some CTCs may pass through the filter or microcolumn gaps, potentially resulting in the loss of some CTCs that overlap with white blood cells. Summary of the Invention

[0006] The purpose of the present invention is to provide a tumor cell separation and capture microfluidic chip and a method of use to solve the above problems.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] Tumor cell separation and capture microfluidic chip, including:

[0009] a first microfluidic chip, wherein a first microchannel is defined in the first microfluidic chip, an inlet of the first microchannel is connected to the outside, and a first microcolumn array is disposed in the first microchannel;

[0010] a second microfluidic chip, wherein a second microchannel is defined in the second microfluidic chip, an inlet of the second microchannel is connected to an outlet of the first microchannel, and a second microcolumn array is disposed in the second microchannel;

[0011] A third microfluidic chip is provided in the third microfluidic chip, wherein the inlet of the third microchannel is connected to the outlet of the second microchannel, a second baffle is provided in the third microchannel, a third microcolumn array is provided on the side of the second baffle close to the inlet of the third microchannel, a curved channel is provided on the side of the second baffle away from the inlet of the third microchannel, an injection port is provided on the sidewall of the second baffle, the outlet of the third microcolumn array and the inlet of the curved channel are connected through the injection port, the curved channel is fixed in the third microchannel, and the outlet of the curved channel is connected to the outside world.

[0012] Preferably, the first micro-pillar array includes two symmetrically arranged baffles, which are fixedly connected between the top wall and the bottom wall of the first microchannel. A plurality of arc-shaped micro-pillar arrays arranged at equal intervals along the length direction of the first microchannel are fixedly connected between the top wall and the bottom wall of the first microchannel. The plurality of arc-shaped micro-pillar arrays are located between the two baffles. The lengths of the plurality of arc-shaped micro-pillar arrays increase sequentially from the entrance to the exit of the first microchannel. The arc-shaped micro-pillar array includes a plurality of blade micro-pillar groups arranged at equal intervals along the length direction. The blade micro-pillar groups include a plurality of blade micro-pillars arranged at equal intervals circumferentially. The blade micro-pillars are fixedly connected between the top wall and the bottom wall of the first microchannel.

[0013] Preferably, the second micropillar array includes several diamond micropillar arrays arranged at equal intervals along the length direction of the second microchannel, several of the diamond micropillar arrays are located between the two opposite inner walls of the second microchannel, the diamond micropillar array includes two groups of small diamond micropillar groups arranged in parallel above and below, the small diamond micropillar groups include several small diamond micropillars arranged at equal intervals along the width direction of the second microchannel, the small diamond micropillars corresponding to the upper and lower parts are staggered, at least one large diamond micropillar is arranged between several of the small diamond micropillars, several of the large diamond micropillars are arranged at equal intervals, the large diamond micropillar is located at the center of several of the small diamond micropillars, and the small diamond micropillars and large diamond micropillars are fixedly connected between the top wall and the bottom wall of the second microchannel.

[0014] Preferably, the third microcolumn array includes a third baffle fixedly connected between the top wall and the bottom wall of the third microchannel and several leaf-vein-shaped microcolumn arrays, a gap is left between the third baffle and the sample inlet, the middle parts of several leaf-vein-shaped microcolumn arrays all intersect on the straight line where the third baffle is located, several leaf-vein-shaped microcolumn arrays are arranged at equal intervals along the length direction of the third microchannel, the leaf-vein-shaped microcolumn array includes several star-shaped microcolumns, several of the star-shaped microcolumns are arranged at equal intervals along the length direction of the leaf-vein-shaped microcolumn array, and the star-shaped microcolumns are fixedly connected between the top wall and the bottom wall of the third microchannel.

[0015] Preferably, the inner side wall of the curved channel is provided with a serrated microcolumn array, the serrated microcolumn array includes a plurality of serrated microcolumns fixedly connected, the plurality of serrated microcolumns are arranged along the length direction of the curved channel, the plurality of serrated microcolumns are respectively located on the two opposite inner side walls of the curved channel and are staggered, the water-facing surface of the serrated microcolumns is at an angle with the inner side wall of the curved channel, the angle is less than 90°, and the water-facing surface of the serrated microcolumns is provided with a plurality of arc grooves.

[0016] Preferably, the distance between two adjacent blade micro-column groups is 5 μm, and the length of the blade micro-column is 15 μm and the width is 6 μm.

[0017] Preferably, the distance between two adjacent small diamond microcolumns is 5 μm, the long diagonal of the small diamond microcolumn is 40 μm, the wide diagonal of the small diamond microcolumn is 20 μm, the long diagonal of the large diamond microcolumn is 400 μm, and the wide diagonal of the large diamond microcolumn is 200 μm.

[0018] Preferably, the angle between the vein-shaped microcolumn array and the vertical direction is 60°, the distance between two adjacent star-shaped microcolumns is 5 μm, the angle between the sawtooth-shaped microcolumns and the horizontal direction is 45°-60°, and the distance between two adjacent sawtooth-shaped microcolumns on the same side is 100 μm.

[0019] The method for using a microfluidic chip for separating and capturing tumor cells comprises the following steps:

[0020] S1, modifying antibodies in the first microchannel and the first microcolumn array;

[0021] S2, inputting the blood sample into the inlet of the first microchannel;

[0022] S3, the first microfluidic chip, the second microfluidic chip and the third microfluidic chip separate and capture tumor cells;

[0023] S4. Purify and enrich circulating tumor cells.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] By setting up a three-layer chip consisting of a first microfluidic chip, a second microfluidic chip, and a third microfluidic chip, the first microfluidic chip is modified with antibodies, thereby achieving dual capture of affinity and physical properties with high capture efficiency; by setting up a three-layer chip, two different tumor cells expressed in epithelial and mesenchymal forms and invasion distances are separated, and different tumor cells are captured; the spectrum or characteristic spectrum of the tumor cells captured at different positions can be obtained, and the spectral characteristics can be better used to analyze the characteristics of genes and proteins, thereby better studying the patient's condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive work.

[0027] Figure 1 1 is a top view of different microfluidic chips of the present invention;

[0028] Figure 2 for Figure 1 A partial enlarged view of point A in the middle;

[0029] Figure 3 for Figure 1 A partial enlarged view of point B in the middle;

[0030] Figure 4 Schematic diagram of the structure of leaf vein micro-pillar array and star-shaped micro-pillar;

[0031] Figure 5 Schematic diagram of the structure of zigzag micropillar array and zigzag micropillar;

[0032] Figure 6 It is a schematic diagram of the three-dimensional structure of the present invention;

[0033] Figure 7 This is a schematic structural diagram of the aggregation chip of Example 3;

[0034] Figure 8 for Figure 7 A partial enlarged view of point C in the middle.

[0035] Among them, 1. first microfluidic chip; 2. second microfluidic chip; 3. third microfluidic chip; 100. first microcolumn array; 101. leaf microcolumn group; 102. leaf microcolumn group spacing; 103. first baffle; 200. second microcolumn array; 201. small diamond microcolumns; 202. small diamond microcolumn spacing; 203. large diamond microcolumns; 300. third microcolumn array; 301. leaf vein microcolumn array; 302. curved channel; 303. serrated microcolumn array; 304. star-shaped microcolumns; 305. star-shaped microcolumn spacing; 306. serrated microcolumns; 307. arc groove; 308. second baffle; 309. third baffle; 310. injection port; 4. aggregation chip; 400. circular microfluidic microcolumn array; 401. leaflet microcolumns; 402. dumbbell-shaped microcolumns. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0038] Example 1

[0039] Reference Figures 1 to 6 As shown, the present invention provides a microfluidic chip for separating and capturing tumor cells, comprising:

[0040] A first microfluidic chip 1 is provided with a first microchannel, the inlet of which is in communication with the outside, and a first microcolumn array 100 is provided in the first microchannel;

[0041] A second microfluidic chip 2 is provided with a second microchannel, the inlet of the second microchannel is connected to the outlet of the first microchannel, and a second microcolumn array 200 is provided in the second microchannel;

[0042] A third microfluidic chip 3 is provided with a third microchannel, the inlet of the third microchannel is connected to the outlet of the second microchannel, a second baffle 308 is provided in the third microchannel, a third micropillar array 300 is provided on the side of the second baffle 308 close to the inlet of the third microchannel, a curved channel 302 is provided on the side of the second baffle 308 away from the inlet of the third microchannel, an injection port 310 is provided on the sidewall of the second baffle 308, the outlet of the third micropillar array 300 and the inlet of the curved channel 302 are connected through the injection port 310, the curved channel 302 is fixed in the third microchannel, and the outlet of the curved channel 302 is connected to the outside world.

[0043] By setting up a three-layer chip consisting of a first microfluidic chip 1, a second microfluidic chip 2, and a third microfluidic chip 3, the first microfluidic chip 1 is a microcolumn array based on affinity capture, modified with anti-epithelial adhesion molecules (anti-EpCAM), and is used to capture and separate tumor cells with epithelial characteristics in whole blood, thereby achieving dual capture of affinity and physical properties with high capture efficiency; by setting up a three-layer chip, two different tumor cells with epithelial expression and mesenchymal expression and invasion distances are separated, and different tumor cells are captured; the spectrum or characteristic spectrum of the tumor cells captured at different positions can be obtained, and the spectral characteristics can be better used to analyze the characteristics of genes and proteins, thereby better studying the patient's condition.

[0044] A further optimized solution is as follows: the first micropillar array 100 includes two symmetrically arranged first baffles 103, which are fixedly connected between the top wall and the bottom wall of the first microchannel. A plurality of arc-shaped micropillar arrays are fixedly connected between the top wall and the bottom wall of the first microchannel. The plurality of arc-shaped micropillar arrays are located between the two first baffles 103. The lengths of the plurality of arc-shaped micropillar arrays increase sequentially from the entrance to the exit of the first microchannel. The arc-shaped micropillar array includes a plurality of blade micropillar groups 101 arranged at equal intervals along the length direction. The blade micropillar groups 101 include a plurality of blade micropillars arranged at equal intervals circumferentially. The blade micropillars are fixedly connected between the top wall and the bottom wall of the first microchannel.

[0045] The number of the leaf micro-columns in the leaf micro-column group 101 of the present invention is 5, and the direction in which the petals and leaves bloom face the direction of fluid flow, which is convenient for capturing circulating tumor cells in the blood.

[0046] Further optimization scheme, the second micropillar array 200 includes several diamond micropillar arrays arranged at equal intervals along the length direction of the second microchannel, several diamond micropillar arrays are located between the two opposite inner walls of the second microchannel, the diamond micropillar array includes two groups of small diamond micropillar groups arranged in parallel above and below, the small diamond micropillar groups include several small diamond micropillars 201 arranged at equal intervals along the width direction of the second microchannel, the upper and lower corresponding small diamond micropillars 201 are staggered, at least one large diamond micropillar 203 is arranged between the several small diamond micropillars 201, several large diamond micropillars 203 are arranged at equal intervals, the large diamond micropillar 203 is located at the center of the several small diamond micropillars 201, the small diamond micropillars 201 and the large diamond micropillars 203 are fixedly connected between the top wall and the bottom wall of the second microchannel.

[0047] Circulating tumor cells in the blood are captured using the distance between adjacent small diamond-shaped micropillars 201. Highly invasive tumor cells with interstitial characteristics are captured at locations farther along the diamond-shaped micropillar array along the direction of fluid flow. Large diamond-shaped micropillars 203 divide the entire capture area into several sections. The upper half of the large diamond-shaped micropillars 203 is triangular. When a blood sample flows to this location, due to the viscosity of the blood molecules, the large diamond-shaped micropillars 203 block the downward flow of the blood sample. Compared to a situation without large diamond-shaped micropillars 203, the blood sample flows slowly down the hypotenuse of the upper triangle. Due to the viscous force between the blood molecules and the hypotenuse, the impact force on the small diamond-shaped micropillars 201 array is reduced. Once captured, CTCs are less likely to flow away from the small diamond-shaped micropillars 201, thereby significantly improving capture efficiency.

[0048] A further optimized solution is provided, in which the third micropillar array 300 includes a third baffle 309 fixedly connected between the top wall and the bottom wall of the third microchannel and a plurality of leaf-vein-shaped micropillar arrays 301. A gap is left between the third baffle 309 and the injection port 310. The middle parts of the plurality of leaf-vein-shaped micropillar arrays 301 all intersect on the straight line where the third baffle 309 is located. The plurality of leaf-vein-shaped micropillar arrays 301 are arranged at equal intervals along the length direction of the third microchannel. The leaf-vein-shaped micropillar array 301 includes a plurality of star-shaped micropillars 304. The plurality of star-shaped micropillars 304 are arranged at equal intervals along the length direction of the leaf-vein-shaped micropillar array 301. The star-shaped micropillars 304 are fixedly connected between the top wall and the bottom wall of the third microchannel.

[0049] Circulating tumor cells in the blood are captured along the direction of fluid flow. Compared to the first two layers, when tumor cells flow to the third microfluidic chip 3, they are primarily circulating tumor cells with highly invasive interstitial characteristics. The third baffle 309 divides the capture area of ​​the entire third micropillar array 300 into two parts. Due to the viscosity of the fluid, the viscous force between the blood sample molecules and the third baffle 309 slows the blood sample flow, thereby improving the capture efficiency of the star-shaped micropillars. The second baffle 308 is set at the entrance of the curved channel 302, and similarly slows the blood sample flow through the viscous force between the blood sample molecules and the second baffle 308.

[0050] In a further optimization, the inner sidewall of the curved channel 302 is provided with a sawtooth micropillar array 303. The sawtooth micropillar array 303 includes a plurality of fixedly connected sawtooth micropillars 306. The sawtooth micropillars 306 are arranged along the length of the curved channel 302 and are located on opposite inner sidewalls of the curved channel 302 in a staggered arrangement. The water-facing surfaces of the sawtooth micropillars 306 form an angle with the inner sidewall of the curved channel 302 that is less than 90°. The water-facing surfaces of the sawtooth micropillars 306 are provided with a plurality of arcuate grooves 307.

[0051] The sawtooth micro-pillars 306 of the present invention have two arc grooves 307 for catching incoming circulating tumor cells. The inclined sawtooth structure can also effectively capture circulating tumor cells.

[0052] According to a further optimized solution, the blade micro-column group spacing 102 between two adjacent blade micro-column groups 101 is 5 μm, the length of the blade micro-column is 15 μm, and the width is 6 μm.

[0053] The number of blade micropillars in a blade micropillar group 101 of the present invention is 5, of which two blade micropillars are symmetrically arranged, the angle formed by the two blade micropillars is 180°, and the distance between them is 8 μm. Another blade micropillar is located at the center of the line connecting the two blade micropillars and is perpendicular to the line connecting the two blade micropillars. The other two micropillars are located on opposite sides of the central blade micropillar and the angle between them is 90°.

[0054] According to a further optimization scheme, the distance between two adjacent small diamond micropillars 201 is 5 μm, the long diagonal of the small diamond micropillar 201 is 40 μm, the wide diagonal of the small diamond micropillar 201 is 20 μm, the long diagonal of the large diamond micropillar 203 is 400 μm, and the wide diagonal of the large diamond micropillar 203 is 200 μm.

[0055] The small diamond micro-pillar spacing 202 between the small diamond micro-pillars 201 is 5 μm, and the small diamond micro-pillar spacing 202 is used to capture circulating tumor cells in the blood.

[0056] According to a further optimization scheme, the angle between the vein-shaped microcolumn array 301 and the vertical direction is 60°, the distance between two adjacent star-shaped microcolumns 304 is 5 μm, the angle between the sawtooth microcolumns 306 and the horizontal direction is 45°-60°, and the distance between two adjacent sawtooth microcolumns 306 on the same side is 100 μm.

[0057] The distance between the star-shaped micropillars is 5 μm. It captures circulating tumor cells in the blood along the direction of fluid flow. Compared with the first two layers, when tumor cells flow to this layer, they are basically circulating tumor cells with strong invasive interstitial characteristics.

[0058] The method for using a microfluidic chip for separating and capturing tumor cells comprises the following steps:

[0059] S1, modifying antibodies in the first microchannel and the first microcolumn array 100;

[0060] S2, inputting the blood sample into the inlet of the first microchannel;

[0061] S3, the first microfluidic chip 1, the second microfluidic chip 2 and the third microfluidic chip 3 separate and capture tumor cells;

[0062] S4. Purify and enrich circulating tumor cells.

[0063] The working process of the present invention is as follows:

[0064] The method for using the tumor cell separation and capture microfluidic chip of the present invention integrates the separation and enrichment of circulating tumor cells (CTCs), is easy to operate, does not rely on tumor cell markers, and is not limited to whether the tumor cells express epithelial adhesion molecule (EpCAM). It is divided into three capture modes:

[0065] Method 1

[0066] For circulating tumor cells expressing EpCAM (epithelial adhesion molecule), the first microchannel and micropillars of the first microfluidic chip 1 are modified with anti-epithelial adhesion molecule (anti-EpCAM). A blood sample is then injected into the inlet of the first microchannel. Circulating tumor cells expressing epithelial cells are captured in the first microfluidic chip 1, while circulating tumor cells expressing invasive, mesenchymal cells are captured in the second and third microfluidic chips 2 and 3, removing blood cells. This highly efficient separation of circulating tumor cells from whole blood also allows for excellent separation and investigation of cancer metastasis, separating epithelial cells with epithelial characteristics from invasive mesenchymal cells.

[0067] Method 2

[0068] The first microfluidic chip 1 is modified with anti-CD45, and a blood sample is input into the inlet of the first microchannel. The large number of white blood cells in the blood are removed in the first microfluidic chip 1. The second microfluidic chip 2 and the third microfluidic chip 3 capture circulating tumor cells (CTCs) in the whole blood and remove the blood cells.

[0069] Method 3

[0070] A whole blood sample from a cancer patient is directly passed through the first microfluidic chip 1, where circulating tumor cells are captured in different layers, capturing the extremely small number of circulating tumor cells in the whole blood. The captured circulating tumor cells are captured in different layers based on their invasiveness, with the most invasive ones being captured at the micropillar array farther along the direction of fluid flow.

[0071] After separating and capturing circulating tumor cells, in order to improve the separation purity, PBS (phosphate buffered saline) is passed into the inlet of the first microfluidic chip 1 at the same optimal flow rate after separation and capture to remove blood cells on the microfluidic chip and improve the separation purity.

[0072] Then, by introducing culture fluid into the microfluidic chip at different time intervals, the captured circulating tumor cells can be cultured and incubated on the microfluidic chip. Doctors can obtain tumor-related gene protein information from the incubated tumor cells and propose corresponding treatment plans.

[0073] Example 2:

[0074] The enrichment of circulating tumor cells in this embodiment differs from that in Example 1 only in that PBS is introduced in reverse through the outlet of the third microfluidic chip 3 to enrich the circulating tumor cells captured on the microfluidic chip. The captured circulating tumor cells are then enriched through the inlet of the first microfluidic chip 1. After centrifugation, the concentrated circulating tumor cells are obtained.

[0075] Example 3

[0076] Reference Figures 7 and 8 The enrichment of circulating tumor cells in this embodiment differs from that in Example 1 only in that PBS is passed through the outlet of the third microfluidic chip 3 to backwash the three-layer microfluidic chip. The captured circulating tumor cells are then enriched in the aggregation chip 4, which comprises seven coaxially arranged circular microfluidic pillar arrays 400. The diameter of the circular microfluidic pillar arrays 400 decreases from the outside to the inside. The diameter of the inner circular microfluidic pillar arrays 400 is 200 μm, and the distance between two adjacent circular microfluidic pillar arrays 400 is 40 μm.

[0077] The circular microfluidic micropillar array 400, located in the inner three layers, is composed of several interconnected lobular micropillars 401. The diameter of the lobular micropillars 401 is 8 μm, and the spacing between adjacent lobular micropillars 401 is 5 μm. The circular microfluidic micropillar array 400, located in the outer four layers, is composed of several interconnected dumbbell-shaped micropillars 402 inclined at 30°. The diameter of the dumbbell-shaped micropillars 402 is 10 μm, and the spacing between adjacent dumbbell-shaped micropillars 402 is 5 μm. This micropillar structure is designed to more effectively and rapidly enrich circulating tumor cells captured by the stacked microfluidic chip in a small area and in a single layer. This allows for better detection of characteristic spectra. By focusing on different locations, the characteristic spectra of the captured circulating tumor cells, namely the gene and protein information of the circulating tumor cells, can be obtained.

[0078] In the description of the present invention, it should be understood that the terms "longitudinal", "transverse", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0079] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A microfluidic chip for separating and capturing tumor cells, characterized in that: include: A first microfluidic chip (1), wherein a first microchannel is provided in the first microfluidic chip (1), an inlet of the first microchannel is connected to the outside, a first microcolumn array (100) is provided in the first microchannel, and antibodies are modified in the first microchannel and the first microcolumn array (100); A second microfluidic chip (2), wherein a second microchannel is provided in the second microfluidic chip (2), an inlet of the second microchannel is connected to an outlet of the first microchannel, and a second microcolumn array (200) is provided in the second microchannel; A third microfluidic chip (3), wherein a third microchannel is provided in the third microfluidic chip (3), the inlet of the third microchannel is communicated with the outlet of the second microchannel, a second baffle (308) is provided in the third microchannel, a third microcolumn array (300) is provided on the side of the second baffle (308) close to the inlet of the third microchannel, a curved channel (302) is provided on the side of the second baffle (308) away from the inlet of the third microchannel, an injection port (310) is provided on the side wall of the second baffle (308), the outlet of the third microcolumn array (300) and the inlet of the curved channel (302) are communicated through the injection port (310), the curved channel (302) is fixed in the third microchannel, and the outlet of the curved channel (302) is communicated with the outside; The first microcolumn array (100) includes two symmetrically arranged first baffles (103), the first baffles (103) are fixedly connected between the top wall and the bottom wall of the first microchannel, and a plurality of arc-shaped microcolumn arrays are fixedly connected between the top wall and the bottom wall of the first microchannel. The plurality of arc-shaped microcolumn arrays are located between the two first baffles (103), and the lengths of the plurality of arc-shaped microcolumn arrays increase from the entrance to the exit of the first microchannel. The arc-shaped microcolumn array includes a plurality of blade microcolumn groups (101) arranged at equal intervals along the length direction, the blade microcolumn group (101) includes a plurality of blade microcolumns arranged at equal intervals in the circumferential direction, and the blade microcolumns are fixedly connected between the top wall and the bottom wall of the first microchannel; the blade microcolumn group spacing (102) between two adjacent blade microcolumn groups (101) is 5 μm, and the length of the blade microcolumn is 15 μm and the width is 6 μm.

2. The tumor cell separation and capture microfluidic chip according to claim 1, characterized in that: The second microcolumn array (200) comprises a plurality of diamond microcolumn arrays arranged at equal intervals along the length direction of the second microchannel, the plurality of diamond microcolumn arrays being located between two opposite inner side walls of the second microchannel, the diamond microcolumn array comprising two groups of small diamond microcolumn groups arranged in parallel up and down, the small diamond microcolumn groups comprising a plurality of small diamond microcolumns (201) arranged at equal intervals along the width direction of the second microchannel, the small diamond microcolumns (201) corresponding to the upper and lower portions being arranged in an alternating manner, at least one large diamond microcolumn (203) being arranged between the plurality of small diamond microcolumns (201), the plurality of large diamond microcolumns (203) being arranged at equal intervals, the large diamond microcolumn (203) being located at the center of the plurality of small diamond microcolumns (201), the small diamond microcolumns (201) and the large diamond microcolumns (203) being fixedly connected between the top wall and the bottom wall of the second microchannel.

3. The tumor cell separation and capture microfluidic chip according to claim 1, characterized in that: The third microcolumn array (300) includes a third baffle (309) fixedly connected between the top wall and the bottom wall of the third microchannel and a plurality of leaf vein-shaped microcolumn arrays (301), a gap is left between the third baffle (309) and the injection port (310), the middle parts of the plurality of leaf vein-shaped microcolumn arrays (301) all intersect on a straight line where the third baffle (309) is located, the plurality of leaf vein-shaped microcolumn arrays (301) are arranged at equal intervals along the length direction of the third microchannel, the leaf vein-shaped microcolumn array (301) includes a plurality of star-shaped microcolumns (304), the plurality of star-shaped microcolumns (304) are arranged at equal intervals along the length direction of the leaf vein-shaped microcolumn array (301), and the star-shaped microcolumns (304) are fixedly connected between the top wall and the bottom wall of the third microchannel.

4. The tumor cell separation and capture microfluidic chip according to claim 3, characterized in that: The inner side wall of the curved channel (302) is provided with a sawtooth microcolumn array (303), the sawtooth microcolumn array (303) includes a plurality of sawtooth microcolumns (306) fixedly connected, the plurality of sawtooth microcolumns (306) are arranged along the length direction of the curved channel (302), the plurality of sawtooth microcolumns (306) are respectively located on two opposite inner side walls of the curved channel (302) and are staggered, the water-facing surface of the sawtooth microcolumns (306) and the inner side wall of the curved channel (302) have an angle, the angle being less than 90°, and the water-facing surface of the sawtooth microcolumns (306) is provided with a plurality of arc grooves (307).

5. The tumor cell separation and capture microfluidic chip according to claim 1, characterized in that: The blade micro-column group spacing (102) between two adjacent blade micro-column groups (101) is 5 μm, and the blade micro-columns have a length of 15 μm and a width of 6 μm.

6. The tumor cell separation and capture microfluidic chip according to claim 2, characterized in that: The distance between two adjacent small diamond-shaped microcolumns (201) is 5 μm, the long diagonal of the small diamond-shaped microcolumns (201) is 40 μm, the wide diagonal of the small diamond-shaped microcolumns (201) is 20 μm, the long diagonal of the large diamond-shaped microcolumns (203) is 400 μm, and the wide diagonal of the large diamond-shaped microcolumns (203) is 200 μm.

7. The tumor cell separation and capture microfluidic chip according to claim 4, characterized in that: The angle between the leaf vein-shaped microcolumn array (301) and the vertical direction is 60°, the distance between two adjacent star-shaped microcolumns (304) is 5 μm, the angle between the sawtooth-shaped microcolumns (306) and the horizontal direction is 45°-60°, and the distance between two adjacent sawtooth-shaped microcolumns (306) on the same side is 100 μm.

8. A method for using a microfluidic chip for separating and capturing tumor cells, based on the microfluidic chip for separating and capturing tumor cells according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1, inputting the blood sample into the inlet of the first microchannel; S2, the first microfluidic chip (1), the second microfluidic chip (2) and the third microfluidic chip (3) separate and capture tumor cells; S3. Purify and enrich circulating tumor cells.

Images