An integrated microfluidic chip for capturing and incubating circulating tumor cells

Abstract

The present invention discloses an integrated microfluidic chip for capturing and incubating circulating tumor cells, belonging to the field of microfluidic technology. The integrated microfluidic chip of the present invention comprises an upper chip, a middle chip and a bottom chip connected in sequence from top to bottom. The first microcolumn array in the upper chip realizes the primary capture of circulating tumor cells. The drainage channel arranged in the middle chip is used to introduce the blood sample from the upper chip into the center of the bottom chip, so that the blood sample flows radially into the U-shaped microcolumn unit for secondary capture. The radially distributed U-shaped microcolumn units make the fluid flux very large, the blood sample flow rate is fast, and the capture rate is high. The present invention overcomes the shortcomings of the prior art microfluidic chips with low flux, insufficient number and purity of enriched tumor cells, resulting in inaccurate detection process. The integrated microfluidic chip provided integrates the capture function and the incubation function, and has high throughput and high capture rate.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics technology, and more specifically, relates to an integrated microfluidics chip for capturing and incubating 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. Almost half of the new cancer cases and more than half of the cancer deaths in 2018 occurred in Asia, because the region has almost 60% of the world's population. The rapid growth of global cancer cases has made how to alleviate and treat tumors a major problem that needs to be solved in the field of life and health of the population in my country and even the world. Traditional treatment methods such as surgical resection, chemotherapy, radiotherapy, immunotherapy, monoclonal antibody therapy or other methods are costly and cause great pain to patients. More than 90% of tumor patients die from tumor metastasis. Circulating tumor cells (CTCs) are malignant tumor cells that fall off from solid tumors or metastatic lesions and enter the peripheral blood circulation. They are a necessary prerequisite for tumor metastasis. And the number of CTCs is closely related to the severity of cancer. A large number means that the condition is aggravated, and a small number means that the condition is slowed down and improved. CTCs detection has great application potential in the discovery of new tumor biomarkers, tumor prognosis judgment and personalized treatment. There are only 1 to 10 CTCs among millions or billions of blood cells. The separation of CTCs must meet the requirements of high capture rate, high purity, high throughput and high activity to meet clinical needs.

[0003] CellSearch is currently the only CTCs detection technology that has been certified by the U.S. Food and Drug Administration (FDA) for clinical use. The CellSearch system achieves the purpose of capturing CTCs by connecting magnetic beads with anti-epithelial cell adhesion molecule antibodies and specific binding of CTCs surface markers. However, the system has the disadvantages of semi-automation, high cost, and low efficiency. Microfluidic chips integrate sample preparation, reaction, separation, detection, etc. on a micrometer-scale chip, automatically completing the entire analysis process. It has the characteristics of small size, low consumption of samples and reagents, and high analysis speed. It is a new type of non-invasive diagnostic tool. The existing capture and separation methods for studying CTCs using microfluidic chips can be divided into two categories: one is to capture and separate cells using the affinity reaction principle of the specific combination of antigen and antibody, and to modify specific antibodies or aptamers such as epithelial adhesion molecules (EpCAM) that can bind to target cell surface antigens on the microchannel or microstructure of the chip. In 2007, Sunitha Nagrath from the Mass General Hospital and Harvard Medical School in the United States reported in Nature that CTCs were captured using a microfluidic chip with microcolumns. On a silicon chip the size of a glass slide, 78,000 microcolumns coated with epithelial adhesion molecule antibodies were arranged. After the patient's blood sample was passed in, it came into contact with the dense support on the surface and collided. The cells efficiently captured by the EpCAM antibody were confirmed as CTCs.

[0004] Before the age of 45, more than 90% of human tumors originate from epithelial cells. However, the expression of EpCAM varies in different tumor cells. And 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. The other type relies on physical properties such as size deformation for capture, using the characteristics of CTCs that are larger than blood cells and less prone to deformation to filter blood cells. For example, Abnova's ClearCell CXSystem is designed and manufactured based on the principle of size deformation. Three cylinders with a spacing of 5 microns form a "claw-shaped" structure, which is arranged repeatedly. Tumor cells are captured, and white blood cells and red blood cells are filtered through deformation.

[0005] In order to improve the capture rate of circulating tumor cells, the inventor has previously designed a double-layer microfluidic chip based on microfluidic technology, with patent application number: 2019111984647, application date: November 29, 2019, and the name of the invention is: a double-layer microfluidic chip for capturing circulating tumor cells. The double-layer microfluidic chip of this scheme includes an upper chip and a lower chip that are correspondingly arranged and interconnected. The upper chip and the lower chip are both provided with microcolumn arrays to achieve multiple captures of tumor cells; and a bowl-shaped structure is also provided in the lower chip to gather tumor cells. When the characteristic spectrum of enriched CTCs is detected by laser, the signal is enhanced, and the tumor type and gene protein spectral characteristics of the tumor patient can be obtained. The capture method 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.

[0006] Although the double-layer chip can combine multiple capture methods and has a high capture rate of circulating tumor cells, the number of circulating tumor cells enriched in the bowl-shaped structure is limited, and because the size of CTCs and white blood cells overlap, some CTCs may pass through the microcolumn gaps. Because they have undergone EMT transformation, the missed CTCs are more malignant, easy to rupture, and have low purity. In addition, the microfluidic chip in this scheme has a low flux. Once the flux is increased, some CTCs may be missed and squeezed through the microcolumn gaps.

[0007] There are only 1 to 10 tumor cells in 1 ml of blood, but there are 10 7 WBCs and 10 9 How to detect every tumor cell in a patient's blood sample is of great significance. This requires a capture rate of nearly 100%, that is, high sensitivity; high purity, that is, the separated CTCs have no blood cells, which is convenient for washing the captured tumor cells from the microfluidic chip for further incubation and biological gene analysis; high throughput, the blood sample used in clinical practice is 7.5ml, while the flow rate of most chips is 1ml / hr, which is far from meeting clinical needs.

[0008] In summary, how to address the shortcomings of the existing technology, such as low flux of microfluidic chips, insufficient number of enriched tumor cells and insufficient purity, which lead to inaccurate detection process, is a technical problem that urgently needs to be solved in the existing technology. Summary of the invention

[0009] 1. Technical problem to be solved by the invention

[0010] The present invention overcomes the shortcomings of the prior art microfluidic chips, such as low flux, insufficient number and purity of enriched tumor cells, which lead to inaccurate detection process, and provides an integrated microfluidic chip for capturing and incubating circulating tumor cells. The upper chip realizes primary capture of circulating tumor cells, and the middle chip introduces the blood sample from the upper chip into the center of the bottom chip, so that the blood sample radially flows into the U-shaped microcolumn unit for secondary capture. The fluid flux is very large, the blood sample flow rate is fast, and the circulating tumor cell capture rate is high.

[0011] 2. Technical solution

[0012] In order to achieve the above object, the technical solution provided by the present invention is:

[0013] An integrated microfluidic chip for capturing and incubating circulating tumor cells of the present invention comprises an upper chip, a middle chip and a bottom chip which are sequentially connected from top to bottom, wherein the upper chip is provided with an upper inlet, a first channel and an upper outlet from the inside to the outside, wherein a first microcolumn array for capturing circulating tumor cells is provided in the first channel, and the minimum spacing between two adjacent microcolumns is no greater than 8 microns; the middle chip is provided with a drainage channel, which is used to introduce a blood sample in the first channel into the bottom chip; the bottom chip is provided with a bottom inlet connected to the drainage channel, a second channel for incubating circulating tumor cells, and a bottom outlet from the inside to the outside, wherein U-shaped microcolumn units radially distributed from the inside to the outside are provided in the second channel, and a microfluidic channel for capturing circulating tumor cells is formed between two adjacent U-shaped microcolumn units, and the width of the microfluidic channel is no greater than 5 microns.

[0014] As a further improvement of the present invention, a circular groove is opened on the bottom chip to form a second channel, and the U-shaped micro-column units are arranged in an array with equal spacing along the circumference of the circular groove. The width of the microfluidic channel formed between two adjacent U-shaped micro-column units is 5 microns.

[0015] As a further improvement of the present invention, the U-shaped microcolumn unit includes at least two relatively arranged elliptical microcolumns and arc-shaped microcolumns, the elliptical microcolumns are arranged relative to the bottom layer inlet, the arc-shaped microcolumns are arranged relative to the bottom layer outlet, and the gap formed between the elliptical microcolumns and the arc-shaped microcolumns is no more than 5 microns.

[0016] As a further improvement of the present invention, the U-shaped micro-column units are distributed in an array with equal spacing along the radial direction of the circular groove.

[0017] As a further improvement of the present invention, a third channel is provided between the second channel and the bottom layer outlet.

[0018] As a further improvement of the present invention, the first channel extends outwardly with the upper layer inlet as the center to form a spiral channel, and the upper layer outlet is connected to the end of the spiral first channel.

[0019] As a further improvement of the present invention, a middle layer inlet and a middle layer outlet for connecting the drainage channel are respectively arranged from the outside to the inside on the middle layer chip, the middle layer inlet is connected to the upper layer outlet, and the middle layer outlet is connected to the bottom layer inlet.

[0020] As a further improvement of the present invention, the drainage channel is a cylindrical channel, and the width of the drainage channel is not less than 100 microns.

[0021] As a further improvement of the present invention, one end of the first microcolumn array located at the upper layer inlet is in contact with the outer side of the first channel, one end of the first microcolumn array located at the upper layer outlet is in contact with the inner side of the first channel, and the first microcolumn array is substantially parallel to the first channel.

[0022] As a further improvement of the present invention, the first micro-column array includes inner micro-columns and outer micro-columns, and the cross-sections of the inner micro-columns and / or the outer micro-columns are polygonal or arc-shaped.

[0023] 3. Beneficial effects

[0024] Compared with the prior art, the technical solution provided by the present invention has the following significant effects:

[0025] (1) An integrated microfluidic chip for capturing and incubating circulating tumor cells of the present invention comprises an upper chip, a middle chip and a bottom chip connected in sequence from top to bottom. The first microcolumn array in the upper chip realizes the primary capture of circulating tumor cells. The drainage channel provided in the middle chip is used to introduce the blood sample from the upper chip into the center of the bottom chip, so that the blood sample flows radially into the U-shaped microcolumn unit for secondary capture. The radially distributed U-shaped microcolumn units make the fluid flux very large, the blood sample flow rate fast, and the circulating tumor cell capture rate high.

[0026] (2) The present invention is an integrated microfluidic chip for capturing and incubating circulating tumor cells. A circular groove is opened on the bottom chip to form a second channel. U-shaped microcolumn units are arranged in an array with equal spacing along the circumference and radial directions of the circular groove. The second channel formed by the circular groove and the radial U-shaped microcolumn units ensure less resistance when the blood sample flows. The blood sample is quickly and efficiently separated in the chip. At the same time, the design of the circular groove also improves the blood sample detection throughput. Combined with the spiral first channel of the upper chip, the test throughput of the microfluidic chip is greatly improved. Using the integrated microfluidic chip of the present invention, 7.5 ml of clinical blood sample detection can be quickly completed.

[0027] (3) The present invention is an integrated microfluidic chip for capturing and incubating circulating tumor cells. The U-shaped microcolumn unit is used to capture and incubate circulating tumor cells. The circulating tumor cells obtained are highly active and the detection results are accurate. The microfluidic chip of the present invention can not only capture circulating tumor cells, but also culture circulating tumor cells after capture, so that the integrated microfluidic chip integrates the capture function and the incubation function. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the structure of the upper chip in the present invention;

[0029] Figure 2 Schematic diagram of the structure of the first microcolumn array (triangular microcolumns) in the present invention;

[0030] Figure 3 Schematic diagram of the structure of the first microcolumn array (square microcolumns and trapezoidal microcolumns) in the present invention;

[0031] Figure 4 Schematic diagram of the structure of the first microcolumn array (semicircular square microcolumns and square microcolumns) in the present invention;

[0032] Figure 5 It is a schematic diagram of the structure of the middle layer chip in the present invention;

[0033] Figure 6 It is a schematic diagram of the structure of the bottom chip in the present invention.

[0034] Reference numerals:

[0035] 100, upper chip; 110, first channel; 111, upper inlet; 112, upper outlet; 113, inside of first channel; 114, outside of first channel; 120, first micro-pillar array; 121, inside micro-pillar; 122, outside micro-pillar;

[0036] 200, middle layer chip; 210, drainage channel; 211, middle layer entrance; 212, middle layer exit;

[0037] 300, bottom chip; 310, second channel; 311, bottom inlet; 312, bottom outlet; 313, third channel; 320, U-shaped microcolumn unit; 321, arc-shaped microcolumn; 322, elliptical microcolumn. DETAILED DESCRIPTION

[0038] 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.

[0039] Example 1

[0040] Combination Figure 1 and Figure 6An integrated microfluidic chip for capturing and incubating circulating tumor cells in this embodiment includes an upper chip 100, a middle chip 200 and a bottom chip 300 connected in sequence from top to bottom. The upper chip 100 is provided with an upper inlet 111, a first channel 110 and an upper outlet 112 from the inside to the outside, respectively. A first microcolumn array 120 for capturing circulating tumor cells is provided in the first channel 110, and the minimum spacing between two adjacent microcolumns is no more than 8 microns; the size of tumor cells is between 10 and 20 μm, that of red blood cells is 4 to 6 μm, and that of white blood cells is 7 to 12 μm. The minimum size between two adjacent microcolumns is smaller than the diameter of the tumor cells, so that tumor cells and some white blood cells can be captured in one capture.

[0041] Preferably, three groups of first channels 110 are provided on the upper chip 100, and each group of first channels 110 is arranged equidistantly and shares an upper inlet 111, the upper inlet 111 is located at the center of the upper chip 100, and the ends of each group of first channels 110 are respectively connected to an upper outlet 112, and the blood sample flows into the three groups of first channels 110 from the upper inlet 111 at the center, and the first micro-column array 120 in the first channel 110 forms a microfluidic channel to achieve one-time capture of circulating tumor cells;

[0042] Specifically, one end of the first microcolumn array 120 located at the upper inlet 111 is in contact with the outer side 114 of the first channel, and one end of the first microcolumn array 120 located at the upper outlet 112 is in contact with the inner side 113 of the first channel. The first microcolumn array 120 is substantially parallel to the first channel 110. The blood sample flows from the inner side 113 of the first channel to the outer side 114 of the first channel and passes through the first microcolumn array 120, thereby being captured by the first microcolumn array 120.

[0043] To facilitate detection, in this embodiment, the three groups of first channels 110 share one detection inlet, and the blood sample flows radially into each first channel 110 from the upper inlet 111, thereby improving the test throughput and detection efficiency.

[0044] Furthermore, combined with Figure 5 In order to make the blood sample in the bottom chip 300 also flow radially into the second channel 310, in this embodiment, a middle chip 200 is arranged between the upper chip 100 and the bottom chip 300, and a drainage channel 210 is arranged on the middle chip 200. The drainage channel 210 is used to introduce the blood sample in the first channel 110 into the bottom chip 300. In this embodiment, a middle inlet 211 and a middle outlet 212 for connecting the drainage channel 210 are arranged on the middle chip 200 from the outside to the inside, respectively. The middle inlet 211 is connected to the upper outlet 112, and the middle outlet 212 is connected to the bottom inlet 311.

[0045] Specifically, in this embodiment, the bottom chip 300 is provided with a bottom inlet 311 connected to the drainage channel 210, a second channel 310 for incubating circulating tumor cells, and a bottom outlet 312 from the inside to the outside. The second channel 310 is provided with U-shaped micro-column units 320 radially distributed from the inside to the outside. A microfluidic channel 320 for capturing circulating tumor cells is formed between two adjacent U-shaped micro-column units, and the width of the microfluidic channel is not more than 5 microns.

[0046] Preferably, in this embodiment, the U-shaped micro-column unit 320 includes at least two elliptical micro-columns 322 arranged opposite to each other, and an arc-shaped micro-column 321, the elliptical micro-column 322 is arranged opposite to the bottom inlet 311, the arc-shaped micro-column 321 is arranged opposite to the bottom outlet 312, and the gap formed between the elliptical micro-column 322 and the arc-shaped micro-column 321 does not exceed 5 microns. Specifically in this embodiment, the arc-shaped micro-column 321 has a length of 30 microns and a width of 8 microns, the major axis of the elliptical micro-column 322 is 10 microns, and the spacing between the elliptical micro-column 322 and the arc-shaped micro-column 321 is 5 microns.

[0047] In the present embodiment, the U-shaped microcolumn unit 320 composed of the elliptical microcolumns 322 and the arc-shaped microcolumns 321 has an opening arranged relative to the bottom inlet 311, and the U-shaped microcolumn units 320 are radially distributed from the inside to the outside, so that the circulating tumor cells in the blood sample can fall into the capture cavity of the U-shaped microcolumn unit 320 in a uniform and dispersed manner. Furthermore, in the subsequent cell incubation process, the culture fluid is introduced into the second channel 310, and the circulating tumor cells can be cultured and grown in the capture cavity of the U-shaped microcolumn unit 320 in a uniform and dispersed manner. The edges of the elliptical microcolumns 322 and the arc-shaped microcolumns 321 are smooth, which can prevent the circulating tumor cells from being punctured, thereby improving the survival rate of the circulating tumor cells.

[0048] In this embodiment, an integrated microfluidic chip for capturing and incubating circulating tumor cells is provided. After the blood sample is captured once by the first microcolumn array 120 in the upper chip 100, it is introduced into the second channel 310 of the bottom chip 300 by the drainage channel 210 on the middle chip 200. The circulating tumor cells are captured again by physical properties in the second channel 310 and are finally captured in the capture cavity of the U-shaped microcolumn unit 320.

[0049] In this embodiment, the U-shaped micro-column units 320 are arranged radially, and the blood sample is injected from the center and flows radially toward the outlet, so that the fluid flux is very large, the blood sample flow rate is fast, the capture rate is high, and the activity of the captured tumor cells is large, and the detection is accurate.

[0050] Furthermore, a catheter can be used to connect the bottom inlet 311 of the bottom chip 300 with the upper inlet 112 of the upper chip 100. The blood sample is captured and enriched by the upper chip 100 and introduced into the bottom chip 300 through the catheter. The circulating tumor cells are captured by the U-shaped microcolumn unit 320. At this time, the catheter is disconnected and the culture medium is introduced from the bottom inlet 311 of the bottom chip 300 to culture the captured tumor cells for molecular genetic analysis and detection, so as to provide treatment plans for tumor patients.

[0051] The integrated microfluidic chip for capturing and incubating circulating tumor cells of the present embodiment can realize the detection of 7.5 ml clinical blood samples. The three first channels 110 of the upper chip 100 are evenly distributed, and the three outlets of the upper chip 100 are the three inlets of the middle chip 200. The three drainage channels 210 with a width of 100 microns of the middle chip 200 lead the blood samples captured by the first layer to the central position and enter the bottom chip 300; the 5-micron microfluidic channel in the bottom chip 300 captures the CTCs leaked from the first layer again.

[0052] The microfluidic chip of this embodiment integrates the capture, separation and enrichment of circulating tumor cells. 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). Among them, the first layer is used to capture CTCs expressing EpCAM, and CTCs with higher malignancy or smaller size are easily missed. Tumor cells undergoing EMT transformation are captured in the third layer.

[0053] The microcolumn array in the microfluidic chip of this embodiment can effectively realize the capture of size. The microfluidic chip can also realize the capture combining affinity and physical size. Specifically, the microchannel is modified with antibodies or aptamers, such as trimethylsiloxane (3mt), coupling agent (GMBS), avidin (Ntra-aviden), and finally modified with anti-epithelial adhesion molecule (anti-EpCAM). When the blood of a tumor patient flows through the microfluidic channel, the surface antigens of the tumor cells bind to the antibodies, the tumor cells are captured, and other blood cells (white blood cells and red blood cells) flow through the channel and flow away from the outlet.

[0054] In addition, a strong magnetic permanent magnet can be placed under the microfluidic chip to generate a strong magnetic field on the surface of the microfluidic chip, and the circulating tumor cells can be coated with magnetic immunomagnetic beads. A blood sample can be introduced at the entrance of the upper chip 100. When the blood sample flows in the channel formed by the first microcolumn array 120, it is captured by the microcolumn array based on size restrictions. On the other hand, the tumor cells connected to the immunomagnetic beads and having magnetism are adsorbed on the bottom of the microfluidic chip under the action of the strong magnetic field. The chip can achieve dual capture of the size and magnetism of tumor cells expressing EpCAM.

[0055] Finally, after the circulating tumor cells are captured for the second time, the magnet is removed to eliminate the magnetic field, and the captured tumor cells are flushed from the outlet with culture fluid or buffer to achieve tumor cell enrichment for the next step of molecular testing.

[0056] Example 2

[0057] The present embodiment is an integrated microfluidic chip for capturing and incubating circulating tumor cells, and its structure is basically the same as that of Example 1. Preferably, in order to achieve high-throughput detection, in the present embodiment, a circular groove is opened on the bottom chip 300 to form a second channel 310, and the U-shaped micro-column units 320 are distributed in an array with equal spacing along the circumference of the circular groove, and the width of the microfluidic channel formed between two adjacent U-shaped micro-column units 320 is 5 microns.

[0058] Furthermore, in this embodiment, the U-shaped micro-column units 320 are distributed in an array with equal spacing along the radial direction of the circular groove, and the U-shaped micro-column units 320 can be used to incubate circulating tumor cells, that is, the microfluidic chip of this embodiment can not only capture circulating tumor cells, but also realize the cultivation of circulating tumor cells after capture, thereby obtaining an integrated microfluidic chip integrating capture and incubation. In the integrated microfluidic chip of the present invention, the second channel 310 formed by the circular groove and the radial U-shaped micro-column units 320 ensure less resistance when the blood sample flows, and the blood sample is quickly and efficiently separated in the chip. At the same time, the design of the circular groove also improves the blood sample detection throughput, and the 7.5ml clinical blood sample detection can be completed quickly.

[0059] Specifically, in this embodiment, the upper inlet 111 of the upper chip 100 is connected to the bottom inlet 311 of the bottom chip 300 by a conduit, the two upper outlets 112 of the upper chip 100 are closed, and phosphate buffer solution PBS is used to wash from the other upper outlet 112 to release the tumor cells captured in the microfluidic channel, and similarly, the circulating tumor cells captured in the other two channels can be released. The released tumor cells flow out from the middle upper inlet 111, flow into the bottom inlet 311 of the bottom chip 300 through the pipeline, and are captured again in the bottom chip 300. The connecting conduit is disconnected, and the culture fluid is injected from the bottom inlet 311 of the bottom chip 300 to realize the culture of the captured tumor cells.

[0060] In addition, in this embodiment, a third channel 313 is provided between the second channel 310 and the bottom outlet 312. After the blood sample is captured by the three-layer chip, it is collected in the third channel 313 and flows out through the bottom outlet 312. The third channel 313 can be used to buffer the resistance generated when the blood sample flows, and also prevent the blood sample from further flowing back. Similarly, in this embodiment, a distance is left between the innermost U-shaped micro-column unit 320 and the bottom inlet 311, and its function is also to prevent the blood sample from being blocked at the bottom inlet 311 and causing backflow.

[0061] Preferably, in order to improve the detection efficiency, the drainage channel 210 in this embodiment is a cylindrical channel, and the width of the drainage channel 210 is not less than 100 microns, so that the blood sample can quickly flow into the bottom chip 300 through the drainage channel 210 for re-capture and further incubation and culture.

[0062] Example 3

[0063] The present embodiment is an integrated microfluidic chip for capturing and incubating circulating tumor cells, and its structure is basically the same as that of Example 1. Furthermore, in the present embodiment, the first channel 110 extends outward with the upper inlet 111 as the center to form a spiral channel, and the upper outlet 112 is connected to the end of the spiral first channel 110.

[0064] Specifically in this embodiment, three groups of spiral channels are nested and evenly distributed on the upper chip 100, and in this embodiment, the first microcolumn array 120 is 400 microns away from the inner side 113 of the first channel and the outer side 114 of the first channel. The spiral first channel 110 improves the test throughput of the microfluidic chip, thereby meeting the clinical 7.5 ml blood sample detection requirements.

[0065] Example 4

[0066] The structure of the integrated microfluidic chip for capturing and incubating circulating tumor cells in this embodiment is basically the same as that in embodiment 1. Figures 2 to 4 In this embodiment, the first micro-pillar array 120 includes an inner micro-pillar 121 and an outer micro-pillar 122 , and the cross-section of the inner micro-pillar 121 and / or the outer micro-pillar 122 is polygonal or arc-shaped.

[0067] In this embodiment, the structures of the inner micro-pillars 121 and the outer micro-pillars 122 may be selected in the following ways:

[0068] like Figure 2 As shown, as another implementation, in this embodiment, the inner microcolumn 121 and the outer microcolumn 122 are both equilateral triangle microcolumns, the minimum spacing between two adjacent equilateral triangle microcolumns is 8 microns, and the side length of the equilateral triangle microcolumn is 100 microns.

[0069] like Figure 3 As shown, in this embodiment, the inner microcolumn 121 is a square microcolumn, and the outer microcolumn 122 is a trapezoidal microcolumn. The spacing between two adjacent square microcolumns is 8 microns, the side length of the square microcolumn is 100 microns, the two bottom sides of the trapezoidal microcolumn are 100 microns and 60 microns respectively, and the height is 50 microns. The width of the gap channel formed by the square microcolumn and the trapezoidal microcolumn is 8 microns. The 8-micron capture gap channel can efficiently capture circulating tumor cells.

[0070] like Figure 4 As shown, as another embodiment, in this embodiment, the inner microcolumn 121 can be a semicircular square microcolumn, the outer microcolumn 122 is a square microcolumn, the spacing of the semicircular square microcolumn array is 8 microns, the spacing between the semicircular square and the square microcolumn is also 8 microns, and the diameters of the square microcolumns and the semicircle are both 100 microns. When the blood sample flux is large and the flow rate is fast, the blood sample flows from the inner side 113 of the first channel to the outer side 114 of the first channel, and the semicircular square microcolumns are arranged on the inner side of the channel, which can prevent tumor cells from being punctured.

[0071] 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. An integrated microfluidic chip for capturing and incubating circulating tumor cells, characterized in that: The invention comprises an upper chip (100), a middle chip (200) and a bottom chip (300) which are connected in sequence from top to bottom, wherein the upper chip (100) is provided with an upper inlet (111), a first channel (110) and an upper outlet (112) from the inside to the outside, respectively, and a first micro-column array (120) for capturing circulating tumor cells is provided in the first channel (110), and the minimum spacing between two adjacent micro-columns is no greater than 8 micrometers; The middle chip (200) is provided with a drainage channel (210), and the drainage channel (210) is used to introduce the blood sample in the first channel (110) into the bottom chip (300); The bottom layer chip (300) is provided with a bottom layer inlet (311) connected to the drainage channel (210), a second channel (310) for incubating circulating tumor cells, and a bottom layer outlet (312) from the inside to the outside, respectively; the second channel (310) is provided with U-shaped micro-column units (320) radially distributed from the inside to the outside, and a micro-flow channel for capturing circulating tumor cells is formed between two adjacent U-shaped micro-column units (320), and the width of the micro-flow channel is not greater than 5 micrometers; A third channel (313) is further provided between the second channel (310) and the bottom layer outlet (312); The first channel (110) extends outwardly with the upper layer inlet (111) as the center to form a spiral channel, and the upper layer outlet (112) is connected to the end of the spiral first channel (110).

2. The integrated microfluidic chip for capturing and incubating circulating tumor cells according to claim 1, characterized in that: A circular groove is formed on the bottom chip (300) to form the second channel (310), the U-shaped micro-column units (320) are distributed in an array with equal spacing along the circumference of the circular groove, and the width of the microfluidic channel formed between two adjacent U-shaped micro-column units (320) is 5 microns.

3. The integrated microfluidic chip for capturing and incubating circulating tumor cells according to claim 2, characterized in that: The U-shaped microcolumn unit (320) comprises at least two elliptical microcolumns (322) arranged opposite to each other, and an arc-shaped microcolumn (321), wherein the elliptical microcolumn (322) is arranged opposite to the bottom layer inlet (311), and the arc-shaped microcolumn (321) is arranged opposite to the bottom layer outlet (312), and a gap formed between the elliptical microcolumn (322) and the arc-shaped microcolumn (321) is no greater than 5 micrometers.

4. The integrated microfluidic chip for capturing and incubating circulating tumor cells according to claim 2, characterized in that: The U-shaped micro-column units (320) are distributed in an array with equal spacing along the radial direction of the circular groove.

5. The integrated microfluidic chip for capturing and incubating circulating tumor cells according to claim 4, characterized in that: A middle layer inlet (211) and a middle layer outlet (212) for communicating with the drainage channel (210) are respectively arranged on the middle layer chip (200) from the outside to the inside; the middle layer inlet (211) is communicated with the upper layer outlet (112), and the middle layer outlet (212) is communicated with the bottom layer inlet (311).

6. The integrated microfluidic chip for capturing and incubating circulating tumor cells according to claim 1, characterized in that: The drainage channel (210) is a cylindrical channel, and the width of the drainage channel (210) is not less than 100 micrometers.

7. An integrated microfluidic chip for capturing and incubating circulating tumor cells according to any one of claims 1 to 6, characterized in that: One end of the first micro-pillar array (120) located at the upper layer inlet (111) is in contact with the outer side (114) of the first channel, and one end of the first micro-pillar array (120) located at the upper layer outlet (112) is in contact with the inner side (113) of the first channel. The first micro-pillar array (120) is substantially parallel to the first channel (110).

8. The integrated microfluidic chip for capturing and incubating circulating tumor cells according to claim 7, characterized in that: The first micro-pillar array (120) comprises inner micro-pillars (121) and outer micro-pillars (122), and the cross-section of the inner micro-pillars (121) and / or the outer micro-pillars (122) is polygonal or arc-shaped.

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