A double-layer microfluidic chip for capturing circulating tumor cells

Abstract

The present invention discloses a double-layer microfluidic chip for capturing circulating tumor cells, and belongs to the field of microfluidic technology. The double-layer microfluidic chip of the present invention comprises an upper chip and a lower chip which are arranged correspondingly and interconnected from top to bottom. Microcolumn arrays are arranged in the upper chip and the lower chip to achieve multiple captures of tumor cells; and a bowl-shaped structure is also arranged in the lower chip to gather tumor cells. When the characteristic spectrum of CTCs is enriched by laser detection, the signal is enhanced, and the tumor type and gene protein spectrum characteristics of the tumor patient can be obtained. The double-layer microfluidic chip of the present invention overcomes the deficiency of low tumor cell capture rate in the prior art, and its structural design is ingenious. The capture method of circulating tumor cells can be combined and selected according to the needs, thereby improving the capture rate of CTCs and avoiding the omission of individual tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics technology, and more specifically, relates to a double-layer microfluidics chip for capturing circulating tumor cells. Background Art

[0002] Cancer is the number one disease threatening human health. In 2018, there were 18.1 million new cancer cases and 9.6 million deaths worldwide, further increasing the global cancer burden. The top three cancer incidences in the world are lung cancer, breast cancer, and colon cancer, and they rank first, fifth, and second in the list of cancer mortality rates. Overall, the incidence and mortality of these three types of cancer account for 1 / 3 of cancers worldwide. 90% of cancer patients die from cancer metastasis, and conquering cancer is a goal that humans have been pursuing. Traditional methods such as resection, chemotherapy, and radiotherapy are costly, cause great pain to patients, and are recurrent. Circulating tumor cells (CTCs) are tumor cells that fall off from primary tumors or regenerated tumors and enter the blood circulation system or lymphatic system. These tumor cells colonize distal tissues, eventually forming cancer metastasis, leading to the death of patients. However, the separation of circulating tumor cells and subsequent molecular biology, gene analysis, etc. are of great significance for tumor detection and diagnosis, tumor progression and metastasis research, and efficacy evaluation. The number of CTCs in the blood circulation system is extremely rare, and it is technically difficult to separate circulating tumor cells from the blood of cancer patients. The number of CTCs is closely related to the severity of cancer. A large number indicates a serious condition, while a small number indicates a mild condition or effective treatment.

[0003] The microchannels of microfluidic chips are comparable in size to cells. It is a new technology with small size, low cost, low reagent consumption and good portability, which is called "liquid biopsy". The existing capture and separation methods of CTCs using microfluidic chips can be roughly divided into three categories:

[0004] One type of cell capture and separation uses the affinity-based principle. This method is based on modifying specific antibodies such as anti-EpCAM such as CTC-chip, or aptamers that can bind to tumor cell surface antigens on the microchannels or microstructures inside the microfluidic chip. The fish-scale chip generated by the micro-vortex of the Herringbone-chip (HB-Chip) changes the micro-column array (CTC-Chip is designed with 78,000 cylindrical micro-columns) into a fish-scale shape, which increases the collision probability between cells and antibody-modified microstructures, and improves the capture efficiency and purity. However, the structure of this microfluidic chip is usually very complex, the modification time is long, the antibodies are expensive, and it depends on the different expressions of EpCAM. When tumor cells undergo epithelial-mesenchymal transition (EMT), the expression of EpCAM in tumor cells decreases. Therefore, relying on this method to capture CTCs may lose some tumor cells that do not express or lowly express EpCAM. This capture method relies on the collision probability of cells and antibody-modified columns. When the flow rate is accelerated, the collision probability of cells and antibody-modified columns is reduced and the interaction time is reduced, and the capture rate decreases. Typical affinity-based microfluidic chip structures include CTC-chip, HB-Chip, a graphene oxide chip, HTMSU, Onco-Bean Chip and a GO-polymer device.

[0005] The other type is based on physical properties such as size deformation (ISET: isolation by size of epithelial tumor cells) for capture, using the characteristics of CTCs that are larger than blood cells and less prone to deformation to filter blood cells. This method is simple to operate, does not need to be too complex in structure, does not require modification, and does not rely on any surface markers. However, since the size of CTCs overlaps with that of leukocytes, some CTCs may pass through the filter or microcolumn gap, resulting in the loss of some CTCs that overlap with the size of leukocytes, especially those with smaller sizes. Because they have undergone EMT transformation, the missed CTCs are more malignant, easy to rupture, low in purity, and prone to clogging. However, compared with affinity or other methods such as two-dimensional electrophoresis, this method is simple and feasible, and is more suitable for clinical applications such as circular spiral chips using inertial separation, CTC aggregation chips, MOA filters, and microfluidic ratchets.

[0006] The third type is magnetic capture, where tumor cells are modified with micromagnetic beads modified with EpCAM antibodies, and the target cells (tumor cells) are coated with micromagnetic beads by mixing and shaking. In this way, the tumor cells are coated with magnetic beads, enlarged in size and magnetic. During capture, a piece of tumor cells with strong magnetism is placed under the microfluidics. The "magnetic" tumor cells connected to the magnetic beads are attracted by the strong magnetism of the magnet and captured under the microfluidic chip, while the non-magnetic blood cells flow away. For example, magnetic-free detection chambers, "Ephesia", quantum dots and magnetic beads for magnetic capture of CTCs, and micromagnetic and microfluidic chips μ-MACS+GASI. In addition, there are also red blood cells that are lysed, white blood cells are "incubated" with magnetic beads, white blood cells are removed by magnetism, and tumor cells are captured by size.

[0007] The sophisticated design of microfluidic chips can realize the application of microfluidic CTCs. Microfluidic CTCs should meet the following requirements: (1) High capture rate: There are only 1-10 tumor cells in 1 ml of blood, but 10 7 WBCs and 10 9 (1) High purity: isolated CTCs are not mixed with other cells such as white blood cells and red blood cells, which is more conducive to detection such as optical detection, identification and counting; (2) High throughput: tumor cells can be separated efficiently and quickly to meet clinical needs; (3) Maintain cell activity: enrichment and activity of tumor cells after capture, so as to facilitate molecular biological gene analysis, thereby determining the patient's condition and treatment plan. Currently, the only one used in clinical practice is Cell Search, which has a low capture rate and is certified by the US FDA. It is limited to breast cancer, colorectal cancer and prostate cancer, but still has the defects of low efficiency and semi-automation.

[0008] The inventor previously designed a tumor cell capture microfluidic chip based on microfluidic technology, with an application date of January 22, 2016 and a patent application number of 2016100441060. The microfluidic chip in this scheme includes a substrate and a microchannel formed in the substrate, the microchannel extends along a smooth curve, and an inlet and an outlet are formed at both ends thereof, a microcolumn array protrudes vertically in the microchannel, the microcolumn array is arranged in an array along the extension direction of the microchannel, one end of the microcolumn array at the inlet is attached to one side wall of the microchannel, and one end of the microcolumn array at the outlet is attached to the other side wall of the microchannel, and a capture cavity with a triangular cross-section is evenly spaced in an array in the middle of the microcolumn array, and the three vertex corners of the capture cavity form three 5-micron gaps respectively for 2-3 captures of a tumor cell. The chip in this scheme combines two capture methods, which is applicable to CTCs that express EpCAM and those that do not express EpCAM. CTCs that do not express or low-express EpCAM and CTCs that are not connected to magnetic beads can be captured using a microcolumn array with a spacing of 5 microns. However, the capture rate of circulating tumor cells in this scheme is not high and needs to be further improved.

[0009] For example, patent application number: 2015102305988, application date: May 8, 2015, the name of the invention is: a double-layer microfluidic chip for tumor cell sorting, which includes a first chip and a second chip located below the first chip, the surface of the first chip has a sample channel, the surface of the second chip opposite to the sample channel has a buffer channel, and the sample channel and the buffer channel are cross-connected. Compared with the CellSearch detection method, the technology of analyzing tumor cells using the double-layer microfluidic chip in this scheme does not require multi-step operations, can be completed in one step continuously, is simple to operate, has a faster detection speed, and has a higher purity of enriched CTCs. However, since the microfluidic chip in this scheme does not use antibodies, but only adopts a single physical scale screening method, it is difficult to achieve accurate capture of circulating tumor cells in the blood. Therefore, in view of the above problems, it is urgent to design a microfluidic chip with high precision and capable of quickly capturing circulating tumor cells to meet the clinical detection of circulating tumor cells in the blood. Summary of the invention

[0010] 1. Technical problem to be solved by the invention

[0011] The present invention provides a double-layer microfluidic chip for capturing circulating tumor cells, which overcomes the deficiency of low tumor cell capture rate in the prior art. The capturing methods of circulating tumor cells can be combined and selected according to the needs, thereby improving the capture rate of CTCs and avoiding the omission of individual tumor cells.

[0012] 2. Technical solution

[0013] In order to achieve the above object, the technical solution provided by the present invention is:

[0014] A double-layer microfluidic chip for capturing circulating tumor cells of the present invention comprises an upper chip and a lower chip which are arranged correspondingly and interconnected with each other, wherein multiple clusters of upper channels are formed in the upper chip extending outward from the center of a circle along a radial direction, an upper inlet is arranged at the center of the circle of the upper chip, and an upper outlet is arranged at the circumference of the circle, the upper inlet and the upper outlet are respectively connected to the upper channel, an upper microcolumn array is arranged in the upper channel, the upper microcolumn array forms a microcolumn channel for capturing circulating tumor cells, and the width of the microcolumn channel is smaller than the diameter of the circulating tumor cells;

[0015] The lower chip extends from the circumference along the radial direction to form a lower channel. The lower chip is provided with a lower inlet at the circumference and a lower outlet at the radius. The lower inlet and the lower outlet are respectively connected to the lower channel, and the upper outlet is connected to the lower inlet.

[0016] A lower microcolumn array is provided in the lower channel, and the lower microcolumn array includes a bowl-shaped structure with a notch. The notch of two adjacent bowl-shaped structures forms a capture cavity for enriching circulating tumor cells. The curvature radius of the notch is not greater than 30 microns, and the width of the bowl bottom channel formed by two adjacent bowl-shaped structures is not greater than 8 microns.

[0017] As a further improvement of the present invention, one end of the upper microcolumn array located at the upper inlet is in contact with the outer side of the upper channel, and one end of the upper microcolumn array located at the upper outlet is in contact with the inner side of the upper channel. The upper microcolumn array is basically parallel to the upper channel, and the blood sample flows along the inner side of the upper channel to the outer side of the upper channel and passes through the upper microcolumn array.

[0018] As a further improvement of the present invention, the upper microcolumn array includes arc microcolumns, rectangular microcolumns and triangular microcolumns arranged in parallel, and the microcolumns are distributed in an array with equal intervals along the extension direction of the upper channel. Adjacent rectangular microcolumns form a first channel along their opposite sides, and the other side of the rectangular microcolumn and the side of the adjacent triangular microcolumn form a second channel. The width of the first channel is greater than the width of the second channel.

[0019] As a further improvement of the present invention, the width of the first channel is 8 microns, and the width of the second channel is 5 microns.

[0020] As a further improvement of the present invention, a cylindrical microcolumn is provided between the triangular microcolumn and the outer side of the upper channel, and a fourth channel of 5 microns is formed between the cylindrical microcolumns.

[0021] As a further improvement of the present invention, the arc-shaped micro-columns are arranged on a side close to the inner side of the upper channel, and the cylindrical micro-columns are arranged on a side close to the outer side of the upper channel.

[0022] As a further improvement of the present invention, the lower microcolumn array includes bowl-shaped structures distributed in an array at equal intervals along the extension direction of the lower channel, and two notches are opened on the side of the bowl-shaped structure relative to the inner side of the lower channel, and the curvature radius of the notch is 30 microns.

[0023] As a further improvement of the present invention, the width of the microfluidic channel between the bowl-shaped structure and the inner side of the lower channel and between the bowl-shaped structure and the outer side of the lower channel is 15 microns, and the width of the bowl bottom channel formed by two adjacent bowl-shaped structures is 8 microns.

[0024] As a further improvement of the present invention, the lower microcolumn array also includes at least two groups of trapezoidal microcolumns arranged along the extension direction of the lower channel, and each group of trapezoidal microcolumns is arrayed from the channel side wall toward the channel center and intersects at the channel center, and a bowl-shaped structure is provided at the intersection.

[0025] As a further improvement of the present invention, the trapezoidal microcolumns are distributed in a non-equidistant array, microcolumn channels are formed between the trapezoidal microcolumns, the width of each microcolumn channel increases successively along the array direction of the trapezoidal microcolumns, and the width of the microcolumn channel is no more than 5 microns.

[0026] 3. Beneficial effects

[0027] Compared with the prior art, the technical solution provided by the present invention has the following significant effects:

[0028] (1) A double-layer microfluidic chip for capturing circulating tumor cells of the present invention comprises an upper chip and a lower chip which are arranged correspondingly and interconnected. Microcolumn arrays are arranged in both the upper chip and the lower chip to achieve multiple captures of tumor cells, thereby improving the capture rate of CTCs and avoiding the omission of individual tumor cells.

[0029] (2) The double-layer microfluidic chip for capturing circulating tumor cells of the present invention has a bowl-shaped structure in the lower chip to enrich tumor cells. When the characteristic spectrum of enriched CTCs is detected by laser, the signal is enhanced, and the tumor type and gene protein spectrum characteristics of the tumor patient can be obtained.

[0030] (3) In a double-layer microfluidic chip for capturing circulating tumor cells of the present invention, the lower microcolumn array further comprises at least two groups of trapezoidal microcolumns arranged along the extension direction of the lower channel, each group of trapezoidal microcolumns are arranged from the side wall of the channel to the center of the channel and intersect at the center of the channel, and a bowl-shaped structure is provided at the intersection; the combination of the trapezoidal microcolumns and the bowl-shaped structure can realize the efficient capture of tumor cells, and the CTCs are flushed into the capture cavity of the bowl-shaped structure by using a buffer solution, so as to improve the purity of the tumor cells to be detected.

[0031] (4) The double-layer microfluidic chip for capturing circulating tumor cells of the present invention has at least six evenly distributed upper channels arranged in the upper chip, and the lower channels are arranged correspondingly. The clustered distribution of the upper channels can effectively avoid various resistances during fluid flow, improve the flux, and quickly and effectively separate circulating tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A top view of the upper chip of the present invention;

[0033] Figure 2 A three-dimensional diagram of the upper chip of the present invention;

[0034] Figure 3 Schematic diagram of the internal structure of the upper channel of the present invention;

[0035] Figure 4 A top view of the lower chip in the present invention;

[0036] Figure 5 Schematic diagram of the internal structure of the lower chip in the present invention;

[0037] Figure 6 A three-dimensional diagram of the lower chip of the present invention;

[0038] Figure 7 Another schematic diagram of the internal structure of the lower chip in the present invention;

[0039] Figure 8 Another stereoscopic view of the lower chip in the present invention;

[0040] Fig. 9 The antibody modification structure diagram of the double-layer microfluidic chip of the present invention;

[0041] Fig.10 This is a photo of circulating tumor cells coated with immunomagnetic beads in the present invention.

[0042] Reference numerals:

[0043] 100, upper passage; 110, inner side of upper passage; 111, outer side of upper passage; 112, upper entrance; 113, upper exit;

[0044] 200, upper microcolumn array; 210, arc-shaped microcolumn; 211, rectangular microcolumn; 212, triangular microcolumn; 213, cylindrical microcolumn; 220, first channel; 221, second channel; 222, third channel; 223, fourth channel;

[0045] 300, lower passage; 310, lower entrance; 311, lower exit; 312, inner side of lower passage; 313, outer side of lower passage;

[0046] 400, lower layer micro-column array; 410, trapezoidal micro-columns; 411, fifth channel; 412, sixth channel; 413, seventh channel; 414, eighth channel; 420, bowl-shaped structure; 421, notch; 422, capture chamber; 423, bowl bottom channel. DETAILED DESCRIPTION

[0047] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with the accompanying drawings and embodiments.

[0048] Example 1

[0049] Combination Figures 1 to 4 A double-layer microfluidic chip for capturing circulating tumor cells in this embodiment includes an upper chip and a lower chip which are arranged correspondingly and interconnected. A plurality of clusters of upper channels 100 are formed in the upper chip extending outward from the center of a circle along a radial direction. An upper inlet 112 is arranged at the center of the circle and an upper outlet 113 is arranged at the circumference of the circle. The upper inlet 112 and the upper outlet 113 are respectively connected to the upper channel 100. An upper micro-pillar array 200 is arranged in the upper channel 100. The upper micro-pillar array 200 forms a micro-pillar channel for capturing circulating tumor cells. The width of the micro-pillar channel is smaller than the diameter of the circulating tumor cells.

[0050] Specifically in this embodiment, the upper microcolumn array 200 includes an arc microcolumn 210, a rectangular microcolumn 211 and a triangular microcolumn 212 arranged in parallel. In this embodiment, the cross-sections of the arc microcolumn 210, the rectangular microcolumn 211 and the triangular microcolumn 212 are arc, rectangular and triangular, respectively. The microcolumns are arranged in an array with equal intervals along the extension direction of the upper channel 100. Adjacent rectangular microcolumns 211 form a first channel 220 along their opposite sides, and the other side of the rectangular microcolumn 211 and the side of the adjacent triangular microcolumn 212 form a second channel 221. The width of the first channel 220 is greater than the width of the second channel 221 and less than the diameter of circulating tumor cells. The first channel 220 and the second channel 221, two capture gap channels, can be used to capture CTCs.

[0051] In order to further sort circulating tumor cells, in this embodiment, the lower chip extends from the circumference along the radial direction to form a lower channel 300, and the lower chip is provided with a lower inlet 310 at the circumference and a lower outlet 311 at the radius. The lower inlet 310 and the lower outlet 311 are respectively connected to the lower channel 300, and the upper outlet 113 is connected to the lower inlet 310 to realize the connection between the two chips; blood cells and missed CTCs enter the lower chip from the upper outlet 113, and CTCs are enriched in the capture cavity 422 formed by the bowl-shaped structure 420.

[0052] Specifically, in this embodiment, a lower micro-column array 400 is provided in the lower channel 300, and the lower micro-column array 400 includes a bowl-shaped structure 420, and a notch 421 is provided on the bowl-shaped structure 420. The notch 421 of two adjacent bowl-shaped structures 420 forms a capture cavity 422 for enriching circulating tumor cells. The radius of curvature of the notch 421 is not greater than 30 microns, and the width of the bowl bottom channel 423 formed by two adjacent bowl-shaped structures 420 is not greater than 8 microns.

[0053] It is worth noting that, in order to control the flow direction of the blood sample, in this embodiment, one end of the upper microcolumn array 200 located at the upper inlet 112 is in contact with the outer side 111 of the upper channel, and one end of the upper microcolumn array 200 located at the upper outlet 113 is in contact with the inner side 110 of the upper channel. The upper microcolumn array 200 is basically parallel to the upper channel 100, so that the blood sample flows along the inner side 110 of the upper channel to the outer side 111 of the upper channel and passes through the upper microcolumn array 200.

[0054] In order to improve the capture rate, this embodiment optimizes the linear channel in the prior art into a surface channel. Specifically, the cross-sections of the arc microcolumn 210, the rectangular microcolumn 211 and the triangular microcolumn 212 in this embodiment are semicircular, square and equilateral triangle, respectively. The diameter of the semicircle, the side length of the rectangle and the side length of the equilateral triangle are 100 microns, 100 microns and 103 microns, respectively, and the plane of the arc microcolumn 210 is connected to the rectangular microcolumn 211. In this embodiment, the width of the first channel 220 is 8 microns, the width of the second channel 221 is 5 microns, and a 5-micron microfluidic channel is also formed between the vertex angles of two adjacent triangular microcolumns 212. In this embodiment, the first channel 220 and the second channel 221 constitute a capture gap channel, which has an increased area and an extended path, forming a labyrinth-like sealing structure, thereby improving the capture rate of CTCs.

[0055] Preferably, in this embodiment, a cylindrical micro-column 213 is further provided between the triangular micro-column 212 and the outer side 111 of the upper channel, and a fourth channel 223 of 5 micrometers is formed between the cylindrical micro-columns 213 .

[0056] Since tumor cells have a high water content, in order to prevent the microcolumns from piercing the tumor cells, the arcuate microcolumns 210 in this embodiment are arranged on the side close to the inner side 110 of the upper channel, and the cylindrical microcolumns 213 are arranged on the side close to the outer side 111 of the upper channel. After the blood sample enters the upper channel 100 through the upper inlet 112, it passes through the microcolumn channel composed of the arcuate microcolumns 210, the rectangular microcolumns 211, the triangular microcolumns 212 and the cylindrical microcolumns 213 in sequence, and the tumor cells are captured in the microcolumn channel.

[0057] It should be noted that the diameter of circulating tumor cells is 10 to 20 microns, the diameter of red blood cells is 4 to 6 microns, and the diameter of white blood cells is 7 to 12 microns. It can be seen that the sizes of tumor cells and white blood cells partially overlap, but white blood cells are easier to deform than tumor cells. The microcolumn channel formed by the upper microcolumn array 200 uses physical size to perform primary screening of cells. In this embodiment, the width of the first channel 220 is 8 microns, and the width of the second channel 221 is 5 microns. When capturing based on physical properties, for circulating tumor cells that are slightly larger and less deformable, the 5-micron and 8-micron capture gap channels are used to capture circulating tumor cells; blood cells that are slightly smaller and more deformable flow away through the gap channels; after capture, rinse with PBS 1-3 times to improve the purity of capture.

[0058] The above double-layer microfluidic chip is used to capture circulating tumor cells. The capture methods can be divided into four types:

[0059] (1) Capture test of circulating tumor cells expressing and not expressing EpCAM: For circulating tumor cells expressing EpCAM (epithelial adhesion molecule), various antibodies such as trimethylsiloxane (3mt), coupling agent (GMBS), avidin (Ntra-aviden) are modified in the microfluidic channel, and finally modified with anti-epithelial adhesion molecule (anti-EpCAM) or modified aptamer (aptamer); when the diluted patient blood sample flows through the microfluidic chip, the antigens of the tumor cells bind to the antibodies and the tumor cells are captured; at the same time, the microcolumn gap based on the physical size plays a role, and the larger tumor cells are captured, while the slightly smaller blood cells flow through the upper microcolumn array 200. This is a capture method that combines affinity and size.

[0060] Combination Fig. 9 , Fig. 9 This is a diagram of antibody modification based on affinity in a microfluidic chip. In this example, the microfluidic channel was first modified with 4% trimethylsiloxane (3mt, 3-mercaptopropyl trimethoxysilane, dissolved in alcohol) for 45 minutes, then treated with 1μM coupling agent GMBS (the coupling agent Ny-maleimidobutyryloxysuccinimide ester), and then treated with 10μg ml -1 Neutravidin was treated at room temperature for 30 min, and then 10 μg ml -1 Antibody modification of microfluidic channels was achieved by overnight treatment with anti-epithelial adhesion molecule (biotinylated EpCAM) at 4°C.

[0061] (2) Circulating tumor cells are coated with magnetic immunomagnetic beads, and a strong permanent magnet is placed under the microfluidic chip to generate a strong magnetic field in the microfluidic chip. When the blood sample is injected into the upper inlet 112, under the action of the strong magnetic field, the blood sample flows from the inner side 110 of the upper channel to the outer side 111 of the upper channel. Blood cells such as white blood cells and red blood cells can drill through the upper microcolumn array 200 due to their small size and easy deformation. The magnetic CTCs are attracted to the bottom surface of the microfluidic chip by the strong magnetic field and adhere to the chip. After the CTCs are captured, they are rinsed with phosphate buffered saline (PBS) to improve the purity of the capture; the magnet is removed to eliminate the magnetism, and the upper inlet 112 is rinsed with culture fluid or buffer to flush the tumor cells captured in the microfluidic channel to achieve enrichment.

[0062] (3) For tumor cells that do not express EpCAM or express low levels of EpCAM, the cells are not connected to the immunomagnetic beads and are captured directly using their size and deformation.

[0063] (4) CTCs are captured and enriched in the capture cavity 422 formed by the bowl-shaped structure 420 in the lower chip. As the number of CTCs enriched increases, the characteristic spectral signal of the enriched CTCs detected by laser is enhanced, and the tumor type and gene protein spectral characteristics of the tumor patient can be obtained.

[0064] In summary, the double-layer microfluidic chip for capturing circulating tumor cells in this embodiment has an ingenious structural design, and the capturing methods of circulating tumor cells can be combined and selected according to needs, thereby improving the capture rate of CTCs and avoiding the omission of individual tumor cells.

[0065] Example 2

[0066] Combination Figure 5 and Figure 6 , Figure 5 : is a schematic diagram of the internal structure of the lower chip. A double-layer microfluidic chip for capturing circulating tumor cells in this embodiment has a structure that is basically the same as that in Example 1. Furthermore, in this embodiment, the lower microcolumn array 400 includes bowl-shaped structures 420 that are evenly spaced in an array along the extension direction of the lower channel 300. The bowl-shaped structure 420 has two notches 421 on the side relative to the inner side 312 of the lower channel. The curvature radius of the notch 421 is 30 microns. The notches 421 of two adjacent bowl-shaped structures 420 form a capture cavity 422 for enriching circulating tumor cells. The bowl-shaped structure 420 is basically parallel to the lower channel 300. The microfluidic channels between the bowl-shaped structure 420 and the inner side 312 of the lower channel and between the bowl-shaped structure 420 and the outer side 313 of the lower channel are 15 microns, and the bowl bottom channel 423 between two adjacent bowl-shaped structures 420 is 8 microns.

[0067] Tumor cells missed in the upper chip can be captured again through the bowl-shaped structure 420. Specifically, the blood sample enters the lower channel 300 from the lower inlet 310, and larger tumor cells flow into the capture chamber 422 and are captured in the bowl bottom channel 423. Even if some tumor cells pass through the microfluidic channel between the bowl-shaped structure 420 and the inner side 312 of the lower channel, they will be captured again by the array-distributed bowl-shaped structure 420, while other blood cells, such as larger white blood cells, flow away from the microfluidic channel between the bowl-shaped structure 420 and the outer side 313 of the lower channel.

[0068] Example 3

[0069] The double-layer microfluidic chip for capturing circulating tumor cells in this embodiment has a structure that is basically the same as that in Example 1. Furthermore, in this embodiment, the lower microcolumn array 400 also includes at least two groups of trapezoidal microcolumns 410 arranged along the extension direction of the lower channel 300. Each group of trapezoidal microcolumns 410 is arranged from the side wall of the channel to the center of the channel and intersects at the center of the channel. A bowl-shaped structure 420 is provided at the intersection.

[0070] Combination Figure 7 and Figure 8 , Figure 7 3 is another schematic diagram of the internal structure of the lower chip. Specifically, in this embodiment, the lower microcolumn array 400 includes four groups of trapezoidal microcolumns 410 arranged along the extension direction of the lower channel 300. Every two groups of trapezoidal microcolumns 410 are arranged from the channel side wall to the channel center and intersect at the channel center to form an arrow-shaped capture channel. Furthermore, the angle formed at the intersection of the two groups of trapezoidal microcolumns 410 is 20° to 40°, for example, it can be 20°, 25°, 30°, 35° and 40°, preferably 30°.

[0071] In this embodiment, the trapezoidal microcolumns 410 are distributed in a non-equidistant array, and microcolumn channels are formed between the trapezoidal microcolumns 410. The width of each microcolumn channel increases in sequence along the array direction of the trapezoidal microcolumns 410, and the width of the microcolumn channel is not greater than 5 microns. Specifically in this embodiment, the trapezoidal microcolumns 410 are right-angled trapezoidal structures, the length of the upper base is 15 microns, and the height is 5 microns. The hypotenuse of each group of trapezoidal microcolumns 410 is on the same extended straight line, and the fifth channel 411, the sixth channel 412, the seventh channel 413 and the eighth channel 414 are formed in sequence between the upper base and the lower base of adjacent trapezoidal microcolumns 410, and the width of each channel is 2 microns, 3 microns, 4 microns and 5 microns, respectively.

[0072] When the blood sample enters the lower inlet 310 of the lower chip from the upper outlet 113 of the upper chip, the micro-column channels that increase in sequence can filter blood cells of smaller size, such as white blood cells and red blood cells. At the same time, each two groups of trapezoidal micro-columns 410 are arrayed from the side wall of the channel to the center of the channel and intersect at the center of the channel. When the buffer flows in, the CTCs can be flushed to the intersection, which is provided with a bowl-shaped structure 420. The CTCs are gathered and enriched in the capture cavity 421 of the bowl-shaped structure 420. The CTCs here are enriched in greater numbers, and the characteristic spectrum detection signal of the tumor cells is enhanced, which is conducive to determining the tumor type and the gene protein characteristics of the tumor cells.

[0073] The double-layer microfluidic chip of this embodiment, whether the microfluidic chip can successfully capture tumor cells depends to a large extent on the efficiency of circulating tumor cells connecting to immunomagnetic beads. If all CTCs can be connected to immunomagnetic beads with a diameter of 4.5 microns, and the more immunomagnetic beads each tumor cell is connected to, the stronger the magnetism of CTCs can be. Under the action of a strong magnetic field, the greater the possibility that CTCs will be attracted to the bottom surface of the microfluidic chip, and at the same time, the size of tumor cells can be enlarged, which can further ensure the success of magnetic capture.

[0074] Combination Fig.10 , Fig.10 This is a picture of colorectal cancer cells connected to magnetic immunomagnetic beads. It can be seen from the picture that all tumor cells are connected to magnetic immunomagnetic beads with a diameter of 4.5 microns, making the tumor cells magnetic and magnifying 4.5 microns-9 microns, thereby achieving the capture method (2) mentioned above. The method of connecting CTCs to magnetic immunomagnetic beads is also very simple to implement. Add 25 microliters of immunomagnetic beads to 1 ml of tumor cell suspension, and mix the cell suspension upside down within half an hour to obtain Fig.10 effect.

[0075] The chip of this embodiment is applicable to CTCs expressing EpCAM and those not expressing EpCAM. The same chip can realize antibody modification and size-binding capture, magnetic capture by immunomagnetic beads, microcolumn gap capture based on physical size, and spectral detection of CTCs enriched at the bottom of the bowl.

[0076] Example 4

[0077] A double-layer microfluidic chip for capturing circulating tumor cells in this embodiment has a structure that is basically the same as that in Example 1. Furthermore, in this embodiment, at least six evenly distributed upper channels 100 are arranged in the upper chip, and the maximum spacing between the upper microcolumn array 200 and the inner side 110 of the upper channel is 400 microns, and the maximum spacing between the upper microcolumn array 200 and the outer side 111 of the upper channel is 600 microns. The channel width of about 1000 microns can realize the cultivation of tumor cells on the chip after capture. More than six petal-shaped upper channels 100 can achieve high throughput, meet the clinical detection requirements of 7.5 ml, and can also realize the detection of multiple blood samples.

[0078] A double-layer microfluidic chip for capturing circulating tumor cells in this embodiment integrates separation and enrichment of circulating tumor cells (CTCs). The chip is easy to operate, does not rely on tumor cell markers, and is not limited to whether tumor cells express epithelial adhesion molecule (EpCAM); the microcolumn array in the microfluidic chip can effectively achieve size capture, dual capture combining size and affinity or size and magnetism, and detection of characteristic spectra; the multi-cluster upper channel 100 can effectively avoid various resistances during fluid flow, so that blood samples can flow smoothly in the microchannel, and the separation of circulating tumor cells can be achieved quickly and effectively.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A double-layer microfluidic chip for capturing circulating tumor cells, characterized in that: The invention comprises an upper chip and a lower chip which are arranged correspondingly and interconnected with each other, wherein a plurality of clusters of upper channels (100) are formed in the upper chip extending outward from the center of a circle along a radial direction, an upper inlet (112) is arranged at the center of the circle of the upper chip, and an upper outlet (113) is arranged at the circumference of the circle, the upper inlet (112) and the upper outlet (113) are respectively connected to the upper channel (100), an upper micro-column array (200) is arranged in the upper channel (100), and the upper micro-column array (200) forms a micro-column channel for capturing circulating tumor cells, and the width of the micro-column channel is smaller than the diameter of the circulating tumor cells; The upper layer micro-column array (200) comprises arc-shaped micro-columns (210), rectangular micro-columns (211) and triangular micro-columns (212) arranged in parallel, the micro-columns being arranged in an array at equal intervals along the extension direction of the upper layer channel (100), adjacent rectangular micro-columns (211) forming a first channel (220) along their opposite sides, the other side of the rectangular micro-columns (211) and the side of the adjacent triangular micro-columns (212) forming a second channel (221), and the width of the first channel (220) being greater than the width of the second channel (221); A cylindrical micro-column (213) is further provided between the triangular micro-column (212) and the outer side (111) of the upper channel; The arc-shaped microcolumn (210) is arranged on a side close to the inner side (110) of the upper channel, and the cylindrical microcolumn (213) is arranged on a side close to the outer side (111) of the upper channel. After the blood sample enters the upper channel (100) through the upper inlet (112), it passes through the microcolumn channel composed of the arc-shaped microcolumn (210), the rectangular microcolumn (211), the triangular microcolumn (212), and the cylindrical microcolumn (213) in sequence. The lower chip extends from the circumference in the radial direction to form a lower channel (300); the lower chip is provided with a lower inlet (310) at the circumference and a lower outlet (311) at the radius; the lower inlet (310) and the lower outlet (311) are respectively connected to the lower channel (300), and the upper outlet (113) is connected to the lower inlet (310); A lower layer micro-pillar array (400) is provided in the lower layer channel (300), the lower layer micro-pillar array (400) comprises a bowl-shaped structure (420), a notch (421) is provided on the bowl-shaped structure (420), the notches (421) of two adjacent bowl-shaped structures (420) form a capture cavity (422) for enriching circulating tumor cells, the radius of curvature of the notch (421) is not greater than 30 micrometers, and the width of the bowl bottom channel (423) formed by the two adjacent bowl-shaped structures (420) is not greater than 8 micrometers; The lower layer micro-column array (400) further comprises at least two groups of trapezoidal micro-columns (410) arranged along the extension direction of the lower layer channel (300), and each group of trapezoidal micro-columns (410) is arranged from the channel side wall towards the channel center and intersects at the channel center, and a bowl-shaped structure (420) is provided at the intersection.

2. A double-layer microfluidic chip for capturing circulating tumor cells according to claim 1, characterized in that: One end of the upper layer micro-column array (200) located at the upper layer inlet (112) is in contact with the outer side (111) of the upper layer channel, and one end of the upper layer micro-column array (200) located at the upper layer outlet (113) is in contact with the inner side (110) of the upper layer channel. The upper layer micro-column array (200) is substantially parallel to the upper layer channel (100), and the blood sample flows along the inner side (110) of the upper layer channel to the outer side (111) of the upper layer channel and passes through the upper layer micro-column array (200).

3. A double-layer microfluidic chip for capturing circulating tumor cells according to claim 2, characterized in that: The width of the first channel (220) is 8 micrometers, and the width of the second channel (221) is 5 micrometers.

4. A double-layer microfluidic chip for capturing circulating tumor cells according to claim 3, characterized in that: A fourth channel (223) of 5 micrometers is formed between the cylindrical micro-pillars (213).

5. A double-layer microfluidic chip for capturing circulating tumor cells according to claim 1, characterized in that: The lower layer micro-pillar array (400) comprises bowl-shaped structures (420) distributed in an array at equal intervals along the extension direction of the lower layer channel (300), and the bowl-shaped structure (420) has two notches (421) on the side opposite to the inner side (312) of the lower layer channel, and the curvature radius of the notches (421) is 30 micrometers.

6. A double-layer microfluidic chip for capturing circulating tumor cells according to claim 5, characterized in that: The microfluidic channel between the bowl-shaped structure (420) and the inner side (312) of the lower channel and between the bowl-shaped structure (420) and the outer side (313) of the lower channel is 15 microns, and the width of the bowl bottom channel (423) formed by two adjacent bowl-shaped structures (420) is 8 microns.

7. A double-layer microfluidic chip for capturing circulating tumor cells according to claim 6, characterized in that: The trapezoidal micro-pillars (410) are distributed in a non-equidistant array, and micro-pillar channels are formed between the trapezoidal micro-pillars (410). The width of each micro-pillar channel increases in sequence along the array direction of the trapezoidal micro-pillars (410), and the width of the micro-pillar channel is no greater than 5 micrometers.

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