A flow channel gradient cell sorting microfluidic chip and sorting method

Abstract

The present invention discloses a flow channel gradient cell sorting microfluidic chip and a sorting method. The microfluidic chip is provided with a microfluidic channel; the microfluidic channel includes a pre-sorting channel and a micro-pillar array sorting channel that are spirally connected end to end. The starting end of the pre-sorting channel is provided with a sample inlet. The micro-pillar array sorting channel is provided with a filter separator. The filter separator divides the micro-pillar array sorting channel into a converging channel and a diverging channel arranged in parallel. The terminal end of the pre-sorting channel is connected to the starting end of the micro-pillar array sorting channel; the filter separator includes a micro-pillar array. The micro-pillar array includes a plurality of micro-pillars arranged in sequence along the length direction of the micro-pillar array sorting channel. A sorting gap is formed between any two adjacent micro-pillars. The present invention promotes the cells to be eliminated to pass through the sorting gap during flow by constructing a converging channel with a gradually decreasing width and a diverging channel with a gradually increasing width, thereby improving the sorting efficiency and the elimination rate of the cells to be eliminated.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic chip cell sorting, and in particular to a flow channel gradient cell sorting microfluidic chip and a sorting method. Background Art

[0002] Circulating tumor cells (CTCs), found in the peripheral blood of cancer patients, are an important, free-living tissue sample with crucial clinical diagnostic value for early cancer diagnosis and prognostic assessment. However, due to their extremely low abundance in the blood, efficient enrichment of sufficient numbers of CTCs is crucial for subsequent detection and analysis. Consequently, methods for sorting and enriching circulating tumor cells are currently attracting significant attention and becoming a hot topic in medical research and treatment.

[0003] As an important prerequisite for cell research, cell sorting is of great value in life science and clinical medicine research. Existing traditional methods include two major categories: one is based on immune recognition characteristics, including fluorescence-activated cell sorting and magnetic-activated cell sorting, and the other is density gradient centrifugation based on the physical properties of cells. However, these methods all have shortcomings such as being time-consuming, costly, and requiring specialized hardware equipment. Microfluidics, which has emerged in recent years, is a technology that uses microchannels to process or manipulate tiny fluids. It has broad application prospects in medical diagnosis, biochemical analysis and other fields, and is also easy to achieve miniaturization and low-cost development of the overall device. Based on this research, various types of microfluidics technologies based on different sorting principles have also been widely used in circulating tumor cell sorting research.

[0004] The more common existing label-free inertial spiral microfluidics based on size differences is the current mainstream of research and has a huge throughput advantage, but the sorting purity is relatively low. This phenomenon is often difficult to meet the needs of clinical testing for applications such as sorting rare cancer cells in the blood that require high-purity sorting effects. The most important interfering factor is that the white blood cells (WBC) contained in the background cells to be separated will overlap with the target cells in a certain range in size, which has a great impact on the separation method based on cell size, reducing the effectiveness of the size-based sorting method and forcing people to make a trade-off between efficiency and selectivity. This type of scenario problem is mainly limited by a single sorting mechanism. For this reason, more and more studies tend to improve the sorting performance by adding multi-dimensional information. Among them, introducing the mechanical properties of the cells themselves to assist is a new development trend.

[0005] Current research results indicate that the mechanical properties of cells, as inherent biophysical markers, are important indicators of changes in their internal structure. They can effectively characterize the physiological state and functional changes of cells, and, combined with other multidimensional cellular information, provide guidance for a variety of applications, including biological research and disease diagnosis. In addition to differences in cell size, CTCs and WBCs also exhibit significant differences in their deformability. CTCs, derived from epithelial cells, typically have larger and more rigid nuclei, resulting in a higher nucleus-to-cytoplasmic ratio (NCR) than WBCs. This difference manifests as CTCs being less deformable than normal blood cells. If the analysis of cell deformation properties can be applied to the design of inertial spiral microfluidic channels for sorting, incorporating cell deformability into the classification criteria and constructing a separation scheme based on a two-dimensional size-deformation mechanism, it is expected that the capture efficiency and purity of the channel can be improved while ensuring good throughput, better meeting clinical application requirements. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a flow channel gradient cell sorting microfluidic chip and a sorting method thereof, aiming to solve the shortcomings of the current traditional inertial spiral channel, such as relatively low capture purity and inability to distinguish well between two types of cells to be sorted with overlapping sizes, and to improve the sorting performance of the system.

[0007] In a first aspect, the present invention provides a flow channel gradient cell sorting microfluidic chip, which is provided with a microfluidic channel; the microfluidic channel includes a pre-sorting channel and a micro-column array sorting channel that are spiral and connected end to end. A sample liquid inlet is provided at the starting end of the pre-sorting channel. A filter separator is provided in the micro-column array sorting channel. The filter separator divides the micro-column array sorting channel into a gradually converging channel and a gradually expanding channel arranged in parallel. The terminal end of the pre-sorting channel is connected to the starting end of the micro-column array sorting channel;

[0008] From inside to outside, the width of the gradually expanding channel gradually decreases, while the width of the gradually converging channel gradually increases. The terminal end of the gradually converging channel is provided with a waste liquid outlet; the terminal end of the gradually expanding channel is provided with a target cell collection port.

[0009] The filter separator comprises a micropillar array. The micropillar array comprises a plurality of micropillars arranged in sequence along the length of the micropillar array sorting channel. A sorting gap is formed between any two adjacent micropillars; the input width of the sorting gap is g; g is determined based on the size of the target cells to be sorted and the cells to be removed, and is set to 100% to 120% of the lower limit of the intersection of the target cell diameter range and the cell diameter range to be removed.

[0010] Preferably, the filter separator further comprises a gradual guide column, which is located at the starting end of the micro-pillar array sorting channel and connected to the inner side wall of the micro-pillar array sorting channel to seal the starting end of the gradually expanding channel.

[0011] Preferably, the convergent channel surrounds the outer side of the divergent channel.

[0012] Preferably, the cross section of the microcolumn is trapezoidal, and the width gradually decreases from the converging channel to the diverging channel, so that the width of the sorting gap gradually increases from the converging channel to the diverging channel. The width of the output section of the sorting gap is 2g to 4g.

[0013] Preferably, the upper base of the microcolumn is 60 μm, the lower base is 40 μm, and the height is 40 μm to 60 μm.

[0014] Preferably, the target cells to be sorted are circulating tumor cells, and the cells to be eliminated are leukocytes; the input end width g of the sorting gap is 10 μm to 12 μm.

[0015] Preferably, the sample liquid inlet is arranged on the inner side of the spiral microfluidic channel; the waste liquid outlet and the target cell collection port are arranged on the outer side of the spiral microfluidic channel.

[0016] Preferably, the number of turns of the micro-column array sorting pipeline is greater than or equal to 4.

[0017] Preferably, the width of the starting end of the tapered channel is 300μm to 350μm, the width of the ending end is 50μm, and the height is 40μm to 60μm; the width of the starting end of the gradually expanding channel is 40μm, the width of the ending end is 290μm to 340μm, and the height is 40μm to 60μm.

[0018] Preferably, the number of turns of the micro-pillar array sorting pipeline is five;

[0019] Preferably, the device further comprises a substrate and a polydimethylsiloxane film bonded to the substrate; the microfluidic channel is arranged on the side of the polydimethylsiloxane film facing the substrate.

[0020] Preferably, the pre-sorting pipeline is a spiral pipeline structure with a rectangular cross-section.

[0021] Preferably, the pre-sorting pipe has one turn, a width of 400 μm to 450 μm, and a height of 40 μm to 60 μm.

[0022] In a second aspect, the present invention provides a cell sorting method, which uses the aforementioned flow channel gradient cell sorting microfluidic chip;

[0023] The cell sorting method comprises the following steps:

[0024] Step 1: obtaining a mixed sample solution containing target cells and cells to be eliminated; the average diameter of the target cells is larger than the average diameter of the cells to be eliminated; and the nuclear-cytoplasmic ratio of the target cells is larger than the nuclear-cytoplasmic ratio of the cells to be eliminated;

[0025] Step 2: introducing a mixed sample solution from the sample inlet; the mixed sample solution flows along the pre-sorting channel to form a preliminary cell equilibrium focusing position;

[0026] Step 3: The mixed sample solution enters the tapered channel from the terminal end of the pre-sorting pipe. The cells in the mixed sample solution initially migrate in the tapered channel, and then, influenced by the micropillar array, the target cells and the cells to be eliminated move laterally in the tapered channel, approaching one side of the micropillar array. The target cells are larger than the size of the sorting gap and are not easily deformed, so they remain in the tapered channel. The cells to be eliminated can pass through the sorting gap directly or after deformation and then pass through the sorting gap to enter the gradually expanding channel.

[0027] Step 4: The cells to be removed enter the gradually expanding channel and are discharged from the waste liquid outlet; the target cells retained in the gradually shrinking channel are discharged from the target cell collection port.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The circulating tumor cell microfluidic chip provided by this invention incorporates cell mechanical properties into inertial sorting based on cell size, using deformation properties to aid in highlighting the deformation differences between two cell types that originally overlap in size. Circulating tumor cells, derived from epithelial cells, tend to have larger and more rigid nuclei, resulting in a higher nucleus-to-cytoplasm ratio. Their deformability is also poorer than that of white blood cells, making them less likely to pass through the sorting gaps of the micropillar array when flowing through the pipeline. This enables the sorting of white blood cells from circulating tumor cells.

[0030] 2. The present invention promotes the cells to be eliminated to pass through the sorting gap during flow by constructing a gradually narrowing channel with a gradually narrowing width and a gradually widening channel with a gradually widening width, thereby improving the sorting efficiency and the elimination rate of the cells to be eliminated.

[0031] 3. The circulating tumor cell microfluidic chip provided by the present invention utilizes a molding process, significantly shortening the manufacturing process and reducing production costs. For circulating tumor cell sorting and enrichment, it offers advantages such as high throughput and capture efficiency, eliminating the need for additional labeling, reducing sample handling and detection procedures, and maintaining good cell viability, minimizing cell loss, and facilitating subsequent detection and analysis.

[0032] 4. The cell sorting microfluidic chip with a gradient flow path provided by this invention is not only suitable for sorting circulating tumor cells and white blood cells, but can also be flexibly applied to sorting other cells of similar size by adjusting relevant parameters. It has good biocompatibility, wide applicability, and flexibility in application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the overall structure of the present invention.

[0034] Figure 2 It is a top view of the structure of the present invention.

[0035] Figure 3 This is a schematic diagram of the structure of the starting end of the micropillar array sorting pipeline in the present invention (ie Figure 2 (a partial schematic diagram of the location of point A in the figure).

[0036] Figure 4 The schematic diagram of the structure of the terminal end of the micropillar array sorting pipeline in the present invention (ie Figure 2 (a partial schematic diagram of the location of point B in the figure).

[0037] Figure 5 This is a physical diagram of the sorting of the microfluidic channel in different numbers of circles from the inside to the outside in an embodiment of the present invention (C1 to C6 in the figure represent the number of circles of the microfluidic channel from the inside to the outside).

[0038] Figure numerals: 1. substrate; 2. polydimethylsiloxane film; 3. microfluidic channel; 4. sample inlet; 5. pre-sorting channel; 6. micro-pillar array sorting channel; 7. waste liquid outlet; 8. target cell collection port; 9. tapered channel; 10. gradient guide column; 11. micro-pillar array; 12. gradually expanding channel. DETAILED DESCRIPTION

[0039] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of the present invention.

[0040] like Figure 1-4As shown, a microfluidic chip for cell sorting with a gradual flow path includes a substrate 1, a polydimethylsiloxane film 2, and a microfluidic channel 3. The microfluidic channel 3 is disposed on the side of the polydimethylsiloxane film 2 facing the substrate 1. The microfluidic channel 3 is spiral-shaped, with a sample inlet 4 at the input end and a waste outlet 7 and a target cell collection port 8 at the output end. The sample inlet 4 is located inside the spiral microfluidic channel 3. The waste outlet 7 and the target cell collection port 8 are located outside the spiral microfluidic channel 3.

[0041] The microfluidic channel 3 includes a pre-sorting channel 5 and a micro-pillar array sorting channel 6, which are connected end to end in a spiral shape. The pre-sorting channel 5 has a rectangular cross-section spiral channel structure. The starting end of the pre-sorting channel 5 is connected to the sample inlet 4.

[0042] The micropillar array sorting channel 6 comprises a converging channel 9, a filter separator, and a diverging channel 12, arranged side by side from the outside in. The converging channel 9, filter separator, and diverging channel 12 all have a spiral shape along their lengths. The filter separator separates the diverging channel 12 from the converging channel 9, allowing only white blood cells to pass through. From the inside out, the width of the diverging channel 12 decreases, while the width of the converging channel 9 increases.

[0043] The filter separator comprises a gradient guide cylinder 10 and a micropillar array 11. The gradient guide cylinder 10 is located at the starting end of the micropillar array sorting channel 6 and is connected to the inner sidewall of the micropillar array sorting channel 6, sealing the starting end of the gradually diverging channel 12. The micropillar array 11 comprises a plurality of trapezoidal micropillars arranged in a spiral line, starting from the gradient guide cylinder 10.

[0044] A sorting gap is formed between any two adjacent trapezoidal micropillars. The cross-section of the trapezoidal micropillars is trapezoidal, and their width gradually decreases from the converging channel 9 to the diverging channel 12, causing the sorting gap to gradually increase in width from the converging channel 9 to the diverging channel 12. The width of the sorting gap at the input end (i.e., the junction between the sorting gap and the converging channel 9) is 10 μm. The sorting gap allows the passage of smaller, more deformable white blood cells while blocking the passage of larger, less deformable circulating tumor cells.

[0045] The terminal end of the pre-sorting channel 5 is connected to the starting end of the converging channel 9. The terminal end of the converging channel 9 is connected to the target cell collection port 8; the terminal end of the diverging channel 12 is connected to the waste liquid outlet 7. During operation, cells discharged from the pre-sorting channel 5 can only enter the converging channel 9; white blood cells within the cells can pass through the sorting gap and enter the diverging channel 12.

[0046] The number of turns of the pre-sorting pipe 5 is one turn, the width is 400 μm to 450 μm, and the height is 40 μm to 60 μm.

[0047] The micropillar array sorting channel 6 has five turns. Within the micropillar array sorting channel 6, the width of the tapered channel 9 at the beginning is 300 to 350 μm, the width at the end is 50 μm, and the height is 40 to 60 μm. The width of the tapered channel 12 at the beginning is 40 μm, the width at the end is 290 to 340 μm, and the height is 40 to 60 μm. The cross-section of the trapezoidal micropillar has an upper base length of 60 μm, a lower base length of 40 μm, and a height of 40 to 60 μm.

[0048] During the sorting process, after the sample solution enters from the sample inlet 4, the cells will be affected by the fluid dynamics characteristics in the microfluidic channel, and will migrate laterally in the microfluidic channel under the combined action of inertial lift and Dean drag until they reach an equilibrium position in the channel. This equilibrium position depends on factors such as cell size and flow rate. At an appropriate flow rate, when the cells enter the pre-sorting channel 5, the circulating tumor cells as the extracted cells and the white blood cells as the cells to be eliminated are focused to different equilibrium positions due to their size differences, and form a preliminary focusing streamline at the end of the pre-sorting channel 5. However, since there are some cells with overlapping sizes between these two types of cells, their equilibrium positions are relatively close. At this time, the focusing position of the circulating tumor cells is closer to the outside of the pre-sorting channel, and the white blood cells are closer to the inside of the pre-sorting channel.

[0049] The initially focused liquid enters the micropillar array sorting channel 6 from the terminal end of the pre-sorting channel 5 to the starting end of the tapered channel 9. The tapered guide pillars 10 isolate the pre-sorting channel 5 from the diverging channel 12, preventing cells from directly entering the diverging channel 12 and potentially affecting sorting performance. Because the micropillar array 11 gradually distributes from its original starting position near the diverging channel 12 toward the terminal end of the tapered channel 9 along the spiral direction of the microfluidic channel 3, the lateral movement of cells flowing from the pre-sorting channel 5 into the micropillar array sorting channel 6 during flow is controlled, ensuring that as many cells as possible have the opportunity to approach the micropillar array 11. The diameter of 12-15 μm is the range of the overlapping size of circulating tumor cells and white blood cells. Among them, the circulating tumor cells originally close to the outside of the pre-sorting channel 5 will still retain the movement trend of the tapered channel 9 after entering the tapered channel 9 of the micro-pillar array sorting channel 6. As the micro-pillar array 11 gradually approaches the tapered channel 9, a small number of circulating tumor cells will move close to the upper bottom surface of the micro-pillar array sorting channel 6. However, since the size of most circulating tumor cells is larger than the array gap, the part of circulating tumor cells that overlap with the size of white blood cells is not easy to deform, and therefore it is difficult for them to enter the 10 μm sorting gap of the micro-pillar array 11; while the white blood cells originally close to the inside of the pre-sorting channel will gradually migrate and move on the upper bottom surface of the sorting array after entering the tapered channel 9, and gradually some white blood cells will enter the gap of the micro-pillar array 11.

[0050] Therefore, in this embodiment, the target cell collection port set at the terminal end of the gradually converging channel 9 can collect circulating tumor cells, and the waste liquid outlet set at the terminal end of the gradually expanding channel 12 can output the white blood cells to be eliminated, thereby realizing the sorting of circulating tumor cells and white blood cells.

[0051] The cell sorting principle of the flow channel gradient cell sorting microfluidic chip is as follows:

[0052] In the application of inertial microfluidic sorting based on the principle of size difference, the sorting performance is easily affected by the sorting cutoff size, especially the phenomenon that the two types of cells to be sorted have size overlap due to reasons such as cell heterogeneity in the actual sample. For sample sorting in such cases, the sorting purity of the microfluidic channel will be greatly reduced, making the sorting method based on size difference ineffective. In order to solve the problem of the failure of a single sorting mechanism when separating samples with overlapping sizes, this embodiment introduces the deformability of cells into the mechanism of cell sorting and combines the feasible deformation of cells with the inertial microfluidic sorting technology based on the principle of size difference. For example, for the sorting of circulating tumor cells and white blood cells, although some cells of the two have size overlap, the two types of cells have significant differences in their deformability. Circulating tumor cells derived from epithelial cells have larger and harder nuclei, and the nucleocytoplasm ratio is greater than that of white blood cells, and therefore show characteristics that are more difficult to deform. By segmenting the pre-sorting channel and the micropillar array sorting channel, cells form initial focused streamlines in the pre-sorting channel based on their size. The unique micropillar array distribution within the micropillar array sorting channel allows the two types of cells to exhibit different motion trajectories based on their deformability, thus achieving separation. Cell deformability provides an additional dimension of information for low-sensitivity applications with overlapping sizes, avoiding tedious steps such as fluorescent labeling. By constructing a microfluidic sorting method with a two-dimensional size-deformation sorting mechanism, this method effectively overcomes the limitations of microfluidic cell sorting based solely on size when dealing with cells with overlapping sizes, while also improving sorting performance.

[0053] When using this flow channel gradient cell sorting microfluidic chip, the sample solution is first injected into the sample inlet 4. In the pre-sorting channel 5, the circulating tumor cells and leukocytes will reach a certain balance under the action of inertial lift and Dean drag, migrate to the focusing position, and form a preliminary sorting distribution. Subsequently, the two types of cells enter the micro-pillar array sorting channel 6. It should be noted that in order to prolong the interaction time between the cells and the surface of the micro-pillar array 11, better highlight the difference in deformation ability between the two types of cells, and increase the possibility of the cells to be eliminated passing through the sorting gap of the micro-pillar array 11, the flow rate control of the chip should be reasonably determined, and the flow rate should not be too high. During the distribution change of the micropillar array 11 along the spiral microfluidic channel 3, the circulating tumor cells whose cell size is larger than the overlapping part of the circulating tumor cells and the white blood cells will flow along the tapered channel 9. After the circulating tumor cells whose size overlaps with the white blood cells approach the upper bottom surface of the micropillar array 11, they will not pass through the sorting gap of the micropillar array 11 due to their poor deformation ability. Finally, the circulating tumor cells will be collected from the target cell collection port 8 at the terminal end of the micropillar array sorting channel 6. After entering the tapered channel 9 of the micropillar array sorting channel 6, the white blood cells to be eliminated will gradually move toward the upper bottom surface of the micropillar array 11. Movement trend, where the leukocytes whose cell size is smaller than the overlapping part of the circulating tumor cells and the leukocytes will directly pass through the micro-pillar array 11 to the gradually expanding channel 12, while the leukocytes whose size overlaps with the circulating tumor cells have stronger deformation ability and can smoothly pass through the sorting gap of the micro-pillar array 11. As the number of turns increases, most of the leukocytes will flow from the gradually shrinking channel 9 through the sorting gap of the micro-pillar array 11 to the gradually expanding channel 12, and finally be output from the waste liquid outlet 7 at the terminal end of the micro-pillar array sorting pipeline 6, thereby realizing the sorting of circulating tumor cells and improving the purity and efficiency of the inertial sorting originally based only on the size principle.

[0054] The preparation process of the flow channel gradient cell sorting microfluidic chip is as follows: a positive mold containing a microfluidic channel 3 structure is prepared using a silicon wafer using a photolithography process; polydimethylsilane is poured into the positive mold and vacuumed and heated to cure, and then the polydimethylsilane film 2 can be peeled off; finally, the polydimethylsilane film 2 is bonded to the cleaned substrate 1 using a plasma cleaning machine to ensure that the two are tightly bonded, thereby completing the preparation of the flow channel gradient cell sorting microfluidic chip.

[0055] The cell sorting method of the flow channel gradient cell sorting microfluidic chip comprises the following steps:

[0056] Step 1: Obtain a mixed sample solution containing the extracted target cells and cells to be eliminated.

[0057] Step 2: introducing a mixed sample solution from the sample inlet 4; the mixed sample solution flows along the pre-sorting channel 5 to form a preliminary cell equilibrium focusing position;

[0058] Step 3: The mixed sample solution enters the micropillar array sorting channel 6 from the terminal end of the pre-sorting channel 5; the cells in the mixed sample solution initially migrate in the tapered channel 9, and are then affected by the layout of the micropillar array 11, which controls the lateral movement range of the cells in the tapered channel 9 so that the cells approach the upper bottom surface of the micropillar array 11; most target cells are larger than the sorting gap size of the micropillar array 11, while a small number of target cells are close to the sorting gap size of the micropillar array 11, but have a high nuclear-cytoplasmic ratio and are not easily deformed, making it difficult for them to pass through the sorting gap of the micropillar array 11 and enter the gradually expanding channel 12; the cells to be rejected are generally smaller than or approximately equal to the sorting gap size of the micropillar array 11 and are easily deformed to pass through the sorting gap and enter the gradually expanding channel 12;

[0059] Step 4: The cells to be removed are output from the waste liquid outlet 7 ; and the target cells are output from the target cell collection port 8 .

[0060] The sorting effect of this embodiment is as follows Figure 5 shown; from Figure 5 As can be seen in the figure, when white blood cells move to the second circle of the micropillar array sorting channel 6, they form a focused streamline on the upper bottom surface of the micropillar array 11. By the third circle of the micropillar array sorting channel 6, the white blood cell focusing phenomenon becomes more pronounced, forming a focused zone with a width of nearly 20 μm. It can be observed that at this point, circulating tumor cells are primarily concentrated in the center of the converging channel, while white blood cells are primarily concentrated on the upper bottom surface of the micropillar array 11 and in the diverging channel. By the fourth circle of the micropillar array sorting channel 6, the number of white blood cells in the converging channel gradually decreases, and the white blood cell focused zone on the upper bottom surface of the micropillar array 11 gradually decreases. White blood cells begin to accumulate in large numbers on the lower bottom surface of the micropillar array 11. By the fifth circle of the micropillar array sorting channel 6, most of the circulating tumor cells remain in the converging channel 9, forming a focused zone of circulating tumor cells near the outside of the converging channel 9, while the majority of white blood cells migrate into the diverging channel 12. Subsequently, circulating tumor cells focused through the terminal end of the converging channel 9 converge to the target cell collection port 8 provided at the output end for output, while white blood cells passing through the terminal end of the diverging channel 12 converge to the waste liquid outlet 7 provided at the output end for output. Thus, it can be seen that this embodiment can achieve the effect of rapid and sufficient separation and collection of target cells and cells to be eliminated.

[0061] The foregoing description is merely an exemplary embodiment of the present invention and does not limit the scope of patent protection of the present invention. Anyone skilled in the art may modify or alter the foregoing embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person skilled in the art without departing from the spirit and technical concepts disclosed herein shall be covered by the claims of the present invention.

Claims

1. A cell sorting microfluidic chip with a gradual flow channel change, characterized by: A microfluidic channel (3) is provided; the microfluidic channel (3) includes a pre-sorting channel (5) and a microcolumn array sorting channel (6) which are spirally connected end to end; a sample liquid inlet (4) is provided at the starting end of the pre-sorting channel (5); a filter separator is provided in the microcolumn array sorting channel (6); the filter separator separates the microcolumn array sorting channel (6) into a tapered channel (9) and a gradually expanding channel (12) arranged in parallel; the terminal end of the pre-sorting channel (5) is connected to the starting end of the microcolumn array sorting channel (6); the tapered channel (9) surrounds the outside of the gradually expanding channel (12); the sample liquid inlet (4) is provided on the inner side of the spiral microfluidic channel (3); the waste liquid outlet (7) and the target cell collection port (8) are provided on the outer side of the spiral microfluidic channel (3); In the direction from inside to outside, the width of the gradually converging channel (9) gradually decreases, and the width of the gradually expanding channel (12) gradually increases; the terminal end of the gradually expanding channel (12) is provided with a waste liquid outlet (7); the terminal end of the gradually converging channel (9) is provided with a target cell collection port (8); The filter separator comprises a microcolumn array (11); the microcolumn array (11) comprises a plurality of microcolumns sequentially arranged in a longitudinal direction of the microcolumn array sorting channel (6); a sorting gap is formed between any two adjacent microcolumns; the input end width of the sorting gap is g; g is determined according to the size of the target cells to be sorted and the cells to be removed, and is set to 100% to 120% of the lower limit of the intersection of the target cell diameter interval and the cell diameter interval to be removed; The filtering separator further comprises a gradual guide column (10); the gradual guide column (10) is located at the starting end of the micro-column array sorting channel (6) and is connected to the inner side wall of the micro-column array sorting channel (6), thereby sealing the starting end of the gradually expanding channel (12).

2. The cell sorting microfluidic chip with gradual flow channel change according to claim 1, characterized in that: The cross section of the microcolumn is trapezoidal, and the width gradually decreases in the direction from the gradually converging channel (9) to the gradually expanding channel (12), so that the width of the sorting gap gradually increases in the direction from the gradually converging channel (9) to the gradually expanding channel (12); the width of the output end of the sorting gap is 2g to 4g.

3. The cell sorting microfluidic chip with gradual flow channel change according to claim 1, characterized in that: The target cells to be sorted are circulating tumor cells, and the cells to be eliminated are white blood cells; the input end width g of the sorting gap is 10 μm to 12 μm.

4. The cell sorting microfluidic chip with gradual flow channel change according to claim 1, characterized in that: The number of turns of the micro-column array sorting pipeline (6) is greater than or equal to 4.

5. The cell sorting microfluidic chip with gradual flow channel change according to claim 1, characterized in that: The width of the starting end of the gradually converging channel (9) is 300 μm to 350 μm, the width of the ending end is 50 μm, and the height is 40 μm to 60 μm; the width of the starting end of the gradually expanding channel (12) is 40 μm, the width of the ending end is 290 μm to 340 μm, and the height is 40 μm to 60 μm.

6. The cell sorting microfluidic chip with gradual flow channel change according to claim 1, characterized in that: It also includes a substrate (1) and a polydimethylsiloxane film (2) bonded to the substrate (1); the microfluidic channel (3) is arranged on the side of the polydimethylsiloxane film (2) facing the substrate (1).

7. A method for sorting circulating tumor cells, characterized in that: A cell sorting microfluidic chip with a gradual flow path change according to any one of claims 1 to 6 is used; The circulating tumor cell sorting method comprises the following steps: Step 1: obtaining a mixed sample solution containing target cells and cells to be eliminated; the average diameter of the target cells is larger than the average diameter of the cells to be eliminated; and the nuclear-cytoplasmic ratio of the target cells is larger than the nuclear-cytoplasmic ratio of the cells to be eliminated; Step 2: introducing a mixed sample solution from the sample inlet (4); the mixed sample solution flows along the pre-sorting channel (5) to form a preliminary cell equilibrium focusing position; Step 3: The mixed sample solution enters the tapered channel (9) from the terminal end of the pre-sorting pipe (5); the cells in the mixed sample solution initially migrate in the tapered channel (9), and are then affected by the micro-pillar array (11), so that the target cells and the cells to be eliminated move laterally in the tapered channel (9) and approach one side of the micro-pillar array (11); the target cells are larger than the size of the sorting gap and are not easily deformed, so that they remain in the tapered channel (9); the cells to be eliminated can directly pass through the sorting gap, or pass through the sorting gap after deformation, and enter the gradually expanding channel (12); Step 4: The cells to be removed enter the gradually expanding channel (12) and are discharged from the waste liquid outlet (7); the target cells retained in the gradually converging channel (9) are discharged from the target cell collection port (8).

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