In vitro analytical diagnostic instrument, microfluidic chip and method for sorting and enriching circulating tumor cells
Patent Information
- Application Number
- CN202110128093.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-01-29
- Publication Date
- 2026-10-09
- Estimated Expiration
- 2041-01-29
AI Technical Summary
其中，传统的微流控芯片的流道循环肿瘤细胞与白细胞容易重叠，不利于循环肿瘤细胞的回收
Smart Images
Figure CN114806799B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell sorting technology, and in particular to an in vitro analysis and diagnostic instrument, a microfluidic chip and method for sorting and enriching circulating tumor cells. Background Technology
[0002] Tumor metastasis occurs when tumor cells detach from the primary or metastatic lesion, circulate in the lymphatic system or peripheral blood, invade distant tissues, and form new tumor foci, ultimately leading to the patient's death. These detached tumor cells are called circulating tumor cells (CTCs). Therefore, by examining the number and types of circulating tumor cells in the blood, the dynamic changes in tumor lesions can be monitored, and the effectiveness of treatment can be assessed. Immunophenotyping and genomic sequencing analyses of isolated circulating tumor cells can identify drug targets, enabling personalized precision treatment. Circulating tumor cells are also extremely important for early tumor screening. When tumors are 1-2 millimeters in size, they are difficult to detect using imaging techniques. However, in the early stages of many cancers, a certain number of circulating tumor cells are present in the blood. For high-risk patients, regular circulating tumor cell testing helps in the early detection and treatment of cancer, preventing disease progression.
[0003] Therefore, sorting and enriching circulating tumor cells (CTCs) from blood is particularly important. However, the concentration of CTCs is extremely low, typically only 1-10 per milliliter of blood, compared to millions of white blood cells and billions of red blood cells per milliliter. Sorting and enriching CTCs is as difficult as finding a needle in a haystack. Techniques for sorting and enriching CTCs can be broadly categorized as: immunomagnetic bead method, density gradient centrifugation, membrane filtration, and microfluidic chip technology. Among these, traditional microfluidic chips often result in overlap between CTCs and white blood cells in the flow channels, hindering the recovery of CTCs. Summary of the Invention
[0004] Therefore, it is necessary to provide an in vitro analysis and diagnostic instrument, a microfluidic chip and method for sorting and enriching circulating tumor cells, which can effectively avoid the overlap of circulating tumor cells and white blood cells and facilitate the recovery of circulating tumor cells.
[0005] A microfluidic chip for sorting and enriching circulating tumor cells includes a functional board, wherein the first side of the functional board is provided with:
[0006] The initial flow channel, connected to the sample inlet, is used to initially aggregate circulating tumor cells and leukocytes in the sample; and
[0007] The fine screening channel is connected to the initial screening channel. A deepening channel is excavated on the side of the fine screening channel away from the accumulation of circulating tumor cells. The deepening channel is arranged along the extension direction of the fine screening channel and the depth of the deepening channel is greater than the depth of the fine screening channel.
[0008] The aforementioned microfluidic chip for sorting and enriching circulating tumor cells (CTCs) dilutes a blood sample and introduces it into the initial screening channel through the sample inlet. Due to the influence of inertial lift and Dean's drag forces, red blood cells, being smaller in diameter, flow chaotically and disorderly in the initial screening channel. White blood cells and CTCs, being larger in diameter, initially aggregate into bands under the balance of forces in the initial screening channel before flowing into the fine screening channel. CTCs have just aggregated into thin bands and are close to the bottom of the fine screening channel's inner wall, while white blood cells have not yet aggregated to the bottom of the channel's inner wall. However, the bands of white blood cells and CTCs are very close together. A deeper channel is created on the side of the fine screening channel away from the CTC aggregation point. The deepened flow channel is set along the extension direction of the fine screening flow channel, and the depth of the deepened flow channel is greater than the depth of the fine screening flow channel. This disrupts the liquid flow state near the outer wall of the fine screening flow channel, changes the inertial lift and drag force, and breaks the original balance. This allows leukocytes to generate a disordered movement state, which in turn makes the leukocytes more evenly distributed in the fine screening flow channel. This avoids the overlap of leukocyte aggregates and circulating tumor cells. Similarly, red blood cells are also more evenly distributed. This ensures that the aggregates of circulating tumor cells are not interfered with, and also prevents leukocytes from aggregating at the bottom of the inner wall of the fine screening flow channel. This facilitates the subsequent separation of circulating tumor cells from leukocytes and is conducive to the recovery of circulating tumor cells.
[0009] In one embodiment, the depth of the deepened channel is 50 μm-200 μm greater than the depth of the fine screening channel; or the depth of the deepened channel is 70 μm-120 μm greater than the depth of the fine screening channel.
[0010] In one embodiment, the fine screening channel is designed such that the ratio of the size of the circulating tumor cells to the hydraulic diameter is less than or equal to 0.5; or the fine screening channel is designed such that the ratio of the size of the circulating tumor cells to the hydraulic diameter is less than or equal to 0.07; or the fine screening channel is designed such that the ratio of the size of the circulating tumor cells to the hydraulic diameter is 0.045 to 0.065; or the fine screening channel is designed such that the ratio of the size of the circulating tumor cells to the hydraulic diameter is 0.05 to 0.06.
[0011] In one embodiment, a first turning channel is further provided on the first side of the functional plate. The radius of curvature of the first turning channel is greater than that of the fine screening channel. The first turning channel communicates with the fine screening channel. The deepening channel extends into the first turning channel and is located on the side away from the accumulation of circulating tumor cells.
[0012] In one embodiment, a removal channel is further provided on the first side of the functional plate. The removal channel is connected to the end of the first turning channel away from the fine screening channel. The deepening channel extends into the removal channel and is located on the side away from the accumulation of circulating tumor cells. A diversion hole penetrating the wall of the removal channel is provided in the removal channel.
[0013] In one embodiment, a second turning channel is further provided on the first side of the functional plate. The radius of curvature of the second turning channel is greater than that of the removal channel. The second turning channel is connected to the end of the removal channel away from the first turning channel. The deepening channel extends into the second turning channel and is located away from the side where circulating tumor cells accumulate.
[0014] In one embodiment, the end of the second turning channel away from the removal channel is provided with an independent recovery channel and a waste liquid channel. The recovery channel is connected to the side of the second turning channel near the accumulation of circulating tumor cells, and the waste liquid channel is connected to the side of the second turning channel near the deepening channel.
[0015] In one embodiment, the ratio of liquid flowing out of the waste liquid channel to liquid flowing out of the recovery channel is 45%-65%: 3%-20%; or the ratio of liquid flowing out of the waste liquid channel to liquid flowing out of the recovery channel is 50%-60%: 5%-10%.
[0016] In one embodiment, the waste liquid flow channel and the recovery flow channel have a reciprocating folding structure.
[0017] In one embodiment, a buffer channel with a reciprocating folding structure is provided on the second side of the functional board, and the buffer channel is connected to the diversion hole.
[0018] In one embodiment, the proportion of liquid flowing out of the buffer channel is 30%-70%; or the proportion of liquid flowing out of the buffer channel is 45%-60%.
[0019] In one embodiment, a blocking member is provided in the removal channel corresponding to the inlet of the diversion hole, and the blocking member is located on the side of the diversion hole away from the deepening channel. The width of the blocking member along the extension direction of the removal channel is greater than the diameter of the diversion hole.
[0020] In one embodiment, a plurality of branch flow holes are sequentially provided along the extension direction of the removal flow channel, a plurality of buffer flow channels with a reciprocating folding structure are provided on the second side surface of the functional plate, and the buffer flow channels are in one-to-one corresponding communication with the branch flow holes; a blocking member is provided in the removal flow channel corresponding to the inlet of the branch flow hole, the blocking member is located on a side of the branch flow hole away from the deepened flow channel, and the width of the blocking member along the extension direction of the removal flow channel is larger than the diameter of the branch flow hole.
[0021] In one embodiment, along the sample flow direction, the distances between the plurality of branch flow holes and the corresponding deepened flow channels gradually increase.
[0022] In one embodiment, along the sample flow direction, the length of the buffer flow channel corresponding to the branch flow hole adjacent to the first turning flow channel is greater than the lengths of other buffer flow channels.
[0023] In one embodiment, the primary selection flow channel comprises an introduction section, a connecting section and a sorting section which are sequentially communicated, one end of the introduction section away from the connecting section is communicated with the sample inlet, and the introduction section is of a reciprocating folding structure.
[0024] In one embodiment, the connecting section comprises a first straight pipe section, a first arc section, a second straight pipe section, a second arc section and a third straight pipe section which are sequentially communicated, the first straight pipe section is connected with one end of the introduction section away from the sample inlet, and the third straight pipe section is connected with the sorting section.
[0025] In one embodiment, the end of the introduction section is arranged in a clockwise direction and connected with the first straight pipe section.
[0026] In one embodiment, the sorting section comprises an engagement section and a main pipe section which are sequentially communicated, the engagement section is connected with the third straight pipe section, the main pipe section is connected with the fine screening flow channel, the width of the engagement section in a direction perpendicular to the extension direction thereof is a, the width of the main pipe section in a direction perpendicular to the extension direction thereof is b, a < b, and the sorting section is an asymmetric wave-shaped channel in the width direction.
[0027] In one embodiment, the fine screening flow channel and the removal flow channel are sinusoidal arc-shaped flow channels.
[0028] In one embodiment, the microfluidic chip for sorting and enriching circulating tumor cells further comprises an upper cover plate and a lower cover plate, a sample inlet is opened on the upper cover plate, the upper cover plate is stacked and connected with the first side surface of the functional plate, a recovery hole, a waste liquid hole and a discharge hole are opened on the lower cover plate, the lower cover plate is stacked and connected with the second side surface of the functional plate, the recovery flow channel is communicated with the recovery hole, the waste liquid flow channel is communicated with the waste liquid hole, and the branch flow hole is communicated with the discharge hole.
[0029] A method for sorting and enriching circulating tumor cells includes the following steps:
[0030] The diluted blood sample is introduced through the sample inlet of the microfluidic chip. After passing through the initial selection channel with a reciprocating folding structure, the circulating tumor cells and white blood cells in the blood sample initially aggregate. The circulating tumor cells aggregate into thin bands and approach the bottom of the inner wall of the initial selection channel, while the white blood cells have not yet aggregated at the bottom of the inner wall of the initial selection channel, and the red blood cells are dispersed in the initial selection channel.
[0031] The blood sample in the initial screening channel is introduced into the fine screening channel. A deeper channel is dug on the side of the fine screening channel away from the aggregation of circulating tumor cells. The circulating tumor cells in the blood sample aggregate into thin bands and are close to the bottom of the inner wall of the fine screening channel, while the white blood cells are in a disordered state and are far away from the bottom of the inner wall of the fine screening channel.
[0032] The aforementioned method for sorting and enriching circulating tumor cells involves diluting a blood sample and introducing it into the initial screening channel through the sample inlet. Due to the influence of inertial lift and Dean's drag forces, red blood cells, being smaller in diameter, flow chaotically and disorderly in the initial screening channel. White blood cells and circulating tumor cells, being larger in diameter, initially aggregate into bands under the balance of forces in the initial screening channel before flowing into the fine screening channel. The circulating tumor cells have just aggregated into thin bands and are close to the bottom of the inner wall of the fine screening channel, while white blood cells have not yet aggregated to the bottom of the inner wall. However, the bands of white blood cells and circulating tumor cells are very close together. To address this, a deeper channel is dug on the side of the fine screening channel away from the aggregation of circulating tumor cells. The deep channel is set along the extension direction of the fine screening channel, and the depth of the deep channel is greater than that of the fine screening channel. This disrupts the liquid flow state near the outer wall of the fine screening channel, changing the inertial lift and drag force, and breaking the original balance. This allows leukocytes to generate a disordered movement state, resulting in a more uniform distribution of leukocytes in the fine screening channel. This avoids the overlap of leukocyte aggregates and circulating tumor cells. Similarly, erythrocytes are also distributed more evenly. This ensures that the aggregates of circulating tumor cells are not interfered with, and also prevents leukocytes from accumulating at the bottom of the inner wall of the fine screening channel. This facilitates the subsequent separation of circulating tumor cells from leukocytes and the recovery of circulating tumor cells.
[0033] In one embodiment, the method for sorting and enriching circulating tumor cells further includes the following steps:
[0034] The blood sample in the fine screening channel is introduced into a first turning channel with a radius of curvature greater than that of the fine screening channel. The circulating tumor cells in the blood sample aggregate into thin bands and move closer to the bottom of the inner wall of the first turning channel.
[0035] The blood sample in the first turning channel is introduced into the removal channel with a diversion hole. The red blood cells and white blood cells in the blood sample flow out of the removal channel through the diversion hole, while the circulating tumor cells in the blood sample aggregate into thin bands and approach the bottom of the inner wall of the removal channel.
[0036] The blood sample remaining after the removal channel is introduced into a second turning channel with a radius of curvature larger than that of the removal channel. The circulating tumor cells in the blood sample aggregate into thin bands and move closer to the bottom of the inner wall of the second turning channel.
[0037] The second turning channel is connected to a recovery channel on the side near the accumulation of circulating tumor cells, and the blood sample on that side is recovered. The other side is connected to a waste liquid channel, and the blood sample on that side is collected.
[0038] The method for sorting and enriching circulating tumor cells further includes the following steps:
[0039] The blood sample recovered from the recovery channel is then reintroduced into the sample inlet of the microfluidic chip, and the aforementioned steps are repeated.
[0040] In one embodiment, the method for sorting and enriching circulating tumor cells includes a blocking member in the removal channel corresponding to the inlet of the diversion hole, wherein the blocking member is located on the side of the diversion hole away from the deepening channel, the width of the blocking member along the extension direction of the removal channel is greater than the diameter of the diversion hole, the proportion of blood samples flowing out through the diversion hole is 30%-70%, and the proportion of blood samples flowing out through the waste liquid channel is 45%-65%.
[0041] An in vitro analytical diagnostic instrument includes a main body and a microfluidic chip for sorting and enriching circulating tumor cells as described above, wherein the microfluidic chip for sorting and enriching circulating tumor cells can be used in conjunction with the main body.
[0042] In one embodiment, the main body is provided with a chip mounting position for mounting the microfluidic chip for sorting and enriching circulating tumor cells, a mixing chamber for mixing the sample, diluent and lysis buffer, and a recovery chamber for recovering circulating tumor cells. The mixing chamber is connected to the sample inlet of the microfluidic chip for sorting and enriching circulating tumor cells, and the recovery chamber is connected to the recovery orifice from which circulating tumor cells flow out of the microfluidic chip for sorting and enriching circulating tumor cells.
[0043] In one embodiment, the in vitro analytical diagnostic instrument further includes a power system and a control system. The control system is used to control the power system to inject the sample, diluent, and lysis buffer into the mixing chamber in a specific ratio and to pass the mixed liquid in the mixing chamber into the microfluidic chip for sorting and enriching circulating tumor cells at a certain flow rate.
[0044] In one embodiment, the in vitro analytical diagnostic instrument further includes a first control valve, and the main body is also provided with a cleaning fluid chamber, a sample chamber, a diluent chamber and a lysis fluid chamber. The cleaning fluid chamber, sample chamber, diluent chamber and lysis fluid chamber are respectively connected to the power system. One end of the first control valve is connected to the outlet of the cleaning fluid chamber, sample chamber, diluent chamber and lysis fluid chamber, and the other end is connected to the mixing chamber.
[0045] In one embodiment, the in vitro analyzer further includes a second control valve, one end of which is connected to the mixing chamber and the other end of which is connected to the recovery chamber, which is also connected to the power system. Attached Figure Description
[0046] Figure 1 This is a schematic diagram of the top cover of a microfluidic chip for sorting and enriching circulating tumor cells in one embodiment.
[0047] Figure 2 This is a schematic diagram of the first side of the functional board of a microfluidic chip for sorting and enriching circulating tumor cells in one embodiment.
[0048] Figure 3 This is a schematic diagram of the second side of the functional board of a microfluidic chip for sorting and enriching circulating tumor cells in one embodiment.
[0049] Figure 4 This is a schematic diagram of the lower cover plate of a microfluidic chip for sorting and enriching circulating tumor cells in one embodiment;
[0050] Figure 5 for Figure 2 A schematic cross-sectional view along section line AA;
[0051] Figure 6 This is a schematic diagram showing the movement of circulating tumor cells, white blood cells, and red blood cells in a blood sample within the inlet and connecting sections of the initial flow channel.
[0052] Figure 7 This is a schematic diagram showing the movement of circulating tumor cells, white blood cells, and red blood cells in the sorting section of the initial selection channel in a blood sample.
[0053] Figure 8 This is a schematic diagram showing the movement of circulating tumor cells, white blood cells, and red blood cells in the first turning channel of a blood sample.
[0054] Figure 9 This diagram illustrates the movement of circulating tumor cells, white blood cells, and red blood cells in a blood sample within the removal flow channel.
[0055] Figure 10This is a schematic diagram showing the movement of circulating tumor cells, white blood cells, and red blood cells in the second turning channel of a blood sample.
[0056] Figure 11 This is a schematic diagram of an in vitro analytical diagnostic instrument in one embodiment.
[0057] Explanation of reference numerals in the attached figures:
[0058] 01. Red blood cells; 02. White blood cells; 03. Circulating tumor cells; 1. Functional plate; 10. Initial selection channel; 11. Inlet port; 110. Inlet section; 112. Straight tube section; 114. Bend section; 116. Long straight tube; 118. Short straight tube; 120. Connecting section; 121. First straight tube section; 122. First arc-shaped section; 123. Second straight tube section; 124. Second arc-shaped section; 125. Third straight tube section; 130. Sorting section; 132. Connecting section; 13. First bend unit; 13a. First sidewall; 13b. Second sidewall; 14. Second bend unit; 14a. Third sidewall; 14b. 1. Fourth sidewall; 134. Main pipe section; 15. Third bend pipe unit; 15a and 15b of the fifth and sixth sidewalls; 16. Fourth bend pipe unit; 16a. Seventh sidewall; 16b. Eighth sidewall; 20. Fine screening channel; 30. Deepened channel; 40. First bend channel; 50. Removal channel; 51. Diverter hole; 52. Blocking element; 60. Second bend channel; 70. Recovery channel; 71. First outlet hole; 80. Waste liquid channel; 81. Second outlet hole; 90. Buffer channel; 2. Upper cover plate; 21. Sample inlet; 3. Lower cover plate; 31. Recovery hole; 32. Waste liquid hole; 33. Discharge hole. Detailed Implementation
[0059] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0060] Please see Figure 1-4 One embodiment provides a microfluidic chip for sorting and enriching circulating tumor cells, including a functional plate 1. Further, the microfluidic chip for sorting and enriching circulating tumor cells also includes an upper cover plate 2 and a lower cover plate 3. The upper cover plate 2 is stacked and connected to a first side of the functional plate 1, and the lower cover plate 3 is stacked and connected to a second side of the functional plate 1. A sample inlet 21 is provided on the upper cover plate 2.
[0061] Reference Figure 2 , 67. A preliminary screening channel 10 and a fine screening channel 20 are provided on the first side of the functional panel 1. The preliminary screening channel 10 is connected to the sample inlet 21 and is used to initially aggregate circulating tumor cells O3 and leukocytes O2 in the sample. The fine screening channel 20 is connected to the preliminary screening channel 10. A deepening channel 30 is provided on the side of the fine screening channel 20 away from the aggregation of circulating tumor cells O3. The deepening channel 30 is arranged along the extension direction of the fine screening channel 20 and the depth of the deepening channel 30 is greater than the depth of the fine screening channel 20.
[0062] After dilution, the blood sample is introduced into the microfluidic chip through the sample inlet 21 of the upper cover plate 2, and flows into the primary selection channel 10 through the sample inlet 11 on the functional plate 1. The sample inlet 11 penetrates the first and second sides of the functional plate 1 and is correspondingly positioned to correspond to the sample inlet 21. Due to the influence of forces such as inertial lift and Dean's drag, refer to... Figure 6 The red blood cells (01) in the sample are smaller in diameter and flow chaotically in the initial selection channel 10, while the white blood cells (02) and circulating tumor cells (03) are larger in diameter and, under the balance of forces in the initial selection channel 10, will initially aggregate into a band before flowing into the fine screening channel 20. Figure 7 At this point, circulating tumor cells 03 have just aggregated into thin bands and are close to the bottom of the inner wall of the fine screening channel 20, while leukocytes 02 have not yet aggregated to the bottom of the inner wall of the channel. However, the bands of leukocytes 02 and circulating tumor cells 03 are very close together. By excavating a deepened channel 30 on the side of the fine screening channel 20 away from the aggregation of circulating tumor cells 03, the deepened channel 30 is set along the extension direction of the fine screening channel 20 and the depth of the deepened channel 30 is greater than the depth of the fine screening channel 20, the liquid flow state near the outer wall of the fine screening channel 20 is disrupted, making the inertial The change in lift and drag disrupts the original balance, causing leukocytes (O2) to move randomly. This results in a more uniform distribution of leukocytes (O2) in the fine screening channel 20, preventing the overlap of leukocyte (O2) aggregates with circulating tumor cells (O3). Similarly, erythrocytes (O1) are also distributed more evenly, ensuring that they do not interfere with the aggregation of circulating tumor cells (O3) and preventing leukocytes (O2) from accumulating at the bottom of the inner wall of the fine screening channel 20. This facilitates the subsequent separation of circulating tumor cells (O3) from leukocytes (O2) and promotes the recovery of circulating tumor cells (O3).
[0063] Reference Figure 2 , 7 In one embodiment, the fine screening channel is an asymmetrical wavy channel with alternating large and small turns. In other embodiments, the fine screening channel may also be a straight channel.
[0064] Specifically, in one embodiment, the fine screening channel is an asymmetrical wavy channel with a rectangular cross-section, the aspect ratio of which varies within the length of the channel to form a single aggregated particle stream.
[0065] Optionally, the aspect ratio of the rectangular cross-section varies between 8 and 30. In another embodiment, the aspect ratio of the rectangular cross-section varies between 11 and 23.
[0066] Optionally, in one embodiment, the fine screening channel is designed such that the ratio of the size of circulating tumor cells to the hydraulic diameter is less than or equal to 0.5. Alternatively, the fine screening channel is designed such that the ratio of the size of circulating tumor cells to the hydraulic diameter is less than or equal to 0.07. Alternatively, the fine screening channel is designed such that the ratio of the size of circulating tumor cells to the hydraulic diameter is 0.045 to 0.065. Alternatively, the fine screening channel is designed such that the ratio of the size of circulating tumor cells to the hydraulic diameter is 0.05 to 0.06. Wherein, the hydraulic diameter Dh is defined as 2wh / (w+h), referring to... Figure 5 , 7 w and h are the width and height of the flow channel.
[0067] Furthermore, referring to Figure 2 , 8 In one embodiment, a first turning channel 40 is further provided on the first side of the functional plate 1. The radius of curvature of the first turning channel 40 is larger than the radius of curvature of the fine screening channel 20, and the first turning channel 40 flows through the fine screening channel 20. Figure 5 The deepened flow channel 30 extends into the first turning flow channel 40 and is located away from the side where circulating tumor cells O3 accumulate. The deepened flow channel 30 makes the distribution of leukocytes O2 more uniform in the first turning flow channel 40, avoiding overlap with circulating tumor cells O3. A first turning flow channel 40 is connected to the fine screening flow channel 20. Due to the large radius of the first turning flow channel 40, the relative advantage of inertial lift is greater. When passing through the first turning flow channel 40, the centrifugal force on the fluid in the middle of the channel is the greatest, causing it to flow towards the outer edge of the channel. The fluid velocity near the channel wall is the lowest, and the centrifugal force is also the smallest, thus being squeezed by the middle fluid. Therefore, under the action of the first turning flow channel 40, the circulating tumor cells O3 can adhere to the bottom of the inner wall of the channel to the maximum extent, preparing for subsequent sorting of circulating tumor cells O3 and removal of leukocytes O2.
[0068] Reference Figure 2 , 5 Optionally, the depth of the deepened flow channel 30 is 50 μm-200 μm greater than the depth of the fine screening flow channel 20. Optionally, the depth of the fine screening flow channel is 100 μm-200 μm. Preferably, the depth of the deepened flow channel 30 is 70 μm-120 μm greater than the depth of the fine screening flow channel 20. Preferably, the depth of the fine screening flow channel is 110 μm-150 μm. Figure 5This is a cross-sectional schematic diagram of the deepening channel 30 within the first turning channel 40. The fine screening channel 20 extends to connect with the first turning channel 40, and a corresponding deepening channel 30 is also formed within the first turning channel 40. The depth difference H between the deepening channel 30 and the first turning channel 40 is 50μm-200μm, preferably 70μm-120μm. This ensures that circulating tumor cells (O3) flow as close as possible to the bottom of the inner wall of either the fine screening channel 20 or the first turning channel 40, while simultaneously disturbing the liquid flow on one side of the deepening channel 30 to prevent overlap between the bands of leukocyte O2 aggregates and circulating tumor cells (O3), which could affect the subsequent recovery rate and purity of circulating tumor cells (O3).
[0069] Reference Figure 2 , 6 In one embodiment, the initial selection channel 10 includes an inlet section 110, a connecting section 120, and a sorting section 130 connected in sequence. The end of the inlet section 110 furthest from the connecting section 120 is connected to the sample inlet 21, and the inlet section 110 has a reciprocating folding structure. (Refer to...) Figure 6 When the sample enters the inlet of the inlet section 110, red blood cells (01), white blood cells (02), and circulating tumor cells (03) are evenly distributed in the flow channel. Red blood cells (01) have a diameter of approximately 6-8 μm, white blood cells (02) have a diameter of approximately 8-12 μm, and circulating tumor cells (03) have a diameter of approximately 20-30 μm. As the liquid flows through several bends in the inlet section 110, white blood cells (02) and circulating tumor cells (03) gradually aggregate, while red blood cells (01) remain evenly distributed. As the liquid flows through the longer connecting section 120, the aggregated band of white blood cells (02) and circulating tumor cells (03) becomes finer. When the liquid flows into the sorting section 130, the aggregated band of white blood cells (02) and circulating tumor cells (03) becomes even finer and closer to the inner wall of the initial sorting flow channel 10. Setting the inlet section 110 as a reciprocating fold structure not only buffers the inflow of blood samples and makes the liquid flow state more stable, but also facilitates the aggregation of circulating tumor cells O3 and white blood cells O2 in this flow channel as the blood sample flows through the inlet section 110 and the connecting section 120.
[0070] Compared to the sorting section 130, the inlet section 110 and the connecting section 120 are elongated flow channels. Optionally, the width of the inlet section 110 and the connecting section 120 is 0.3mm-1.2mm. Preferably, the width of the inlet section 110 and the connecting section 120 is 0.5mm-0.9mm. Optionally, the depth of the inlet section 110 and the connecting section 120 is 0.06mm-0.3mm. Preferably, the depth of the inlet section 110 and the connecting section 120 is 0.1mm-0.2mm. This configuration allows the flow rate of the blood sample entering the initial sorting flow channel 10 to stabilize, while simultaneously allowing circulating tumor cells 03 and leukocytes 02 to initially aggregate into a band, facilitating subsequent sorting of the circulating tumor cells 03.
[0071] Specifically, refer to Figure 6 In one embodiment, the inlet section 110 includes multiple straight pipe sections 112 and multiple bent pipe sections 114. Two adjacent straight pipe sections 112 are connected by a bent pipe section 114. The initial straight pipe section 112 is connected to the sample inlet 21 through the sample inlet 11, and the final bent pipe section 114 is arranged in a clockwise direction and connected to the connecting section 120. This arrangement creates multiple bends in the inlet section 110, gradually stabilizing the blood sample flow and providing a buffering effect. This facilitates the initial aggregation of leukocytes O2 and circulating tumor cells O3, and the processing is easier compared to spiral pipes, allowing for a smaller overall chip size. In other embodiments, to achieve the buffering purpose, the inlet section 110 can also be configured as a reciprocating "S" shape or a spiral shape. The final bent pipe section 114 of the inlet section 110 is arranged in a clockwise direction and connected to the connecting section 120. The main purpose is to ensure that circulating tumor cells O3 always aggregate on the inner wall of the flow channel during subsequent sorting.
[0072] In one embodiment, the straight pipe segment 112 between the initial straight pipe segment 112 and the final curved pipe segment 114 includes alternating long straight pipes 116 and short straight pipes 118. Both ends of the same long straight pipe 116 (e.g., the lower long straight pipe 116) are connected to two short straight pipes 118 via a clockwise curved pipe segment 114. Alternatively, both ends of the same long straight pipe 116 (e.g., the upper long straight pipe 116) are connected to two short straight pipes 118 via a counterclockwise curved pipe segment 114. Clockwise and counterclockwise directions are determined along the sample flow direction. By alternating the long straight pipes 116 and short straight pipes 118, the straight pipe segments 112 are arranged in multiple rows in the direction perpendicular to the sample flow, allowing for a more rational arrangement of the flow channels and facilitating a reduction in the length and width of the chip.
[0073] Furthermore, the long straight pipe 116 and the short straight pipe 118 are arranged parallel to each other along the sample flow direction. Two adjacent long straight pipes 116 and the short straight pipe 118 located between them are spaced apart along the width of the straight pipe section 112. The long straight pipes 116 and the short straight pipes 118 are connected by a 180° bend 114. The long straight pipes 116 and the short straight pipes 118 are arranged parallel to each other, forming three rows along the width of the straight pipe section 112.
[0074] Specifically, refer to Figure 6At the very beginning (the inlet end of the inlet section 110), the straight pipe section 112 is connected to a long straight pipe 116 via clockwise and counterclockwise curved pipe sections 114. At the very end, the straight pipe section 112 is connected to the connecting section 120 via counterclockwise and clockwise curved pipe sections 114. When the sample flows into the connecting section 120, it flows clockwise, which facilitates the initial aggregation of circulating tumor cells O3 into a band on one side of the inner wall of the flow channel.
[0075] Reference Figure 2 , 6 In one embodiment, the connecting segment 120 includes a first straight pipe segment 121, a first arc-shaped segment 122, a second straight pipe segment 123, a second arc-shaped segment 124, and a third straight pipe segment 125 connected in sequence. The first straight pipe segment 121 is connected to the end of the inlet segment 110 away from the sample inlet 21, and the third straight pipe segment 125 is connected to the sorting segment 130. Specifically, the end of the inlet segment 110 is arranged in a clockwise direction and connected to the first straight pipe segment 121, so that the circulating tumor cells O3 always accumulate on the inner wall of the flow channel. The connecting section 120 connects the inlet section 110 and the sorting section 130. The blood sample flows clockwise from the end of the inlet section 110 through the first straight section 121, the first arc-shaped section 122, the second straight section 123, the second arc-shaped section 124, and the third straight section 125. The bands where leukocytes (O2) and circulating tumor cells (O3) accumulate gradually narrow. When the liquid is in the third straight section 125 and about to enter the sorting section 130, the bands where leukocytes (O2) and circulating tumor cells (O3) accumulate become even narrower and closer to the inner wall of the pipe. In one embodiment, the first arc-shaped section 122 is a 90° arc-shaped section arranged clockwise, and the second arc-shaped section 124 is also a 90° arc-shaped section arranged clockwise. The first straight section 121 and the third straight section 125 are spaced vertically, with the third straight section 125 located above the first straight section 121.
[0076] Furthermore, a first region 126 is formed between the delay line of the first straight pipe section 121 and the delay line of the third straight pipe section 125, and the inlet section 110 folds back and forth within the first region. By rationally arranging the positions of each segment of the flow channel, the volume of the chip is fully utilized, which facilitates the miniaturization of the overall chip size and also facilitates the initial aggregation of leukocytes O2 and circulating tumor cells O3 into a band.
[0077] Reference Figure 2 , 7In one embodiment, the sorting section 130 comprises a connecting section 132 and a main pipe section 134 that are sequentially in communication. The connecting section 132 is connected to the third straight pipe section 125, and the main pipe section 134 is connected to the fine screening flow channel 20. The width of the connecting section 132 in a direction perpendicular to its extension direction is a, and the width of the main pipe section 134 in a direction perpendicular to its extension direction is b, wherein a < b. The sorting section 130 is a wavy channel that is asymmetrical in the width direction. In order to avoid that when the blood sample enters the wavy sorting section 130 from the slender connecting section 120, the sudden large increase in the width of the flow channel disturbs the original movement trajectory of circulating tumor cells 03 and prevents destroying the original trajectory of circulating tumor cells 03 moving along the inner wall, the connecting section 132 with a smaller width than the main pipe section 134 is provided to transition to the main pipe section 134, which plays a buffering role. It avoids the situation that circulating tumor cells 03 can only be close to the inner wall of the flow channel to gather into a thin strip after flowing through more wavy main pipe sections 134, and can also shorten the length of the main pipe section 134.
[0078] Reference Figure 7 Specifically, the connecting section 132 comprises alternating first curved pipe units 13 and second curved pipe units 14, wherein the curvature radius of the first curved pipe unit 13 is larger than that of the second curved pipe unit 14. The main pipe section 134 comprises alternating third curved pipe units 15 and fourth curved pipe units 16, wherein the curvature radius of the third curved pipe unit 15 is larger than that of the fourth curved pipe unit 16. The rearmost second curved pipe unit 14 is connected to the foremost third curved pipe unit 15. Wherein a is the width of the first curved pipe unit 13 in the direction perpendicular to its extension direction, and b is the width of the third curved pipe unit 15 in the direction perpendicular to its extension direction. The connecting section 132 is formed by alternately connecting the first curved pipe units 13 with a large curvature radius and the second curved pipe units 14 with a small curvature radius, and the main pipe section 134 is formed by alternately connecting the third curved pipe units 15 with a large curvature radius and the fourth curved pipe units 16 with a small curvature radius. The width of the connecting section 132 in the direction perpendicular to its extension direction mainly refers to the width of the first curved pipe unit 13 in the direction perpendicular to its extension direction, and the width of the main pipe section 134 in the direction perpendicular to its extension direction mainly refers to the width of the third curved pipe unit 15 in the direction perpendicular to its extension direction. According to factors such as cell diameter, flow channel height and width, and liquid flow rate, combined with force analysis of inertial lift force, Dean drag force and other forces, circulating tumor cells 03 are close to the bottom of the inner wall of the main pipe section 134, and white blood cells 02 also gradually approach the bottom of the inner wall of the main pipe section 134. Optionally, b is 0.4 mm to 1.2 mm larger than a. Preferably, b is 0.7 mm to 0.9 mm larger than a.
[0079] Reference Figure 7Furthermore, the first bending unit 13 includes a first sidewall 13a and a second sidewall 13b arranged opposite to each other, the first sidewall 13a and the second sidewall 13b being asymmetrical curved surfaces. The second bending unit 14 includes a third sidewall 14a and a fourth sidewall 14b arranged opposite to each other, the third sidewall 14a and the fourth sidewall 14b being asymmetrical curved surfaces. The third sidewall 14a is connected to the second sidewall 13b, and the fourth sidewall 14b is connected to the first sidewall 13a. The third bending unit 15 includes a fifth sidewall 15a and a sixth sidewall 15b arranged opposite to each other, the fifth sidewall 15a and the sixth sidewall 15b being asymmetrical curved surfaces. The fourth bending unit 16 includes a seventh sidewall 16a and an eighth sidewall 16b arranged opposite to each other, the seventh sidewall 16a and the eighth sidewall 16b being asymmetrical curved surfaces. The fifth sidewall 15a is connected to the eighth sidewall 16b, and the sixth sidewall 15b is connected to the seventh sidewall 16a. The first bend unit 13 and the second bend unit 14 protrude in different directions, the third bend unit 15 and the fourth bend unit 16 protrude in different directions, and the first bend unit 13 and the third bend unit 15 protrude in the same direction. This asymmetric curved surface configuration creates asymmetric inertial aggregation, causing circulating tumor cells to focus at a stable position in the cross-section of the flow channel (bottom of the inner wall of the flow channel) to form a band, resulting in focused flow downstream.
[0080] Furthermore, the radius of curvature of the first sidewall 13a is smaller than that of the second sidewall 13b, and the radius of curvature of the fifth sidewall 15a is larger than that of the sixth sidewall 15b. This makes the distance between the first sidewall 13a and the second sidewall 13b smaller than the distance between the fifth sidewall 15a and the sixth sidewall 15b, thus achieving buffering of the fluid flow from the connecting section 132 to the main pipe section 134. Furthermore, the radius of curvature of the third sidewall 14a is smaller than that of the fourth sidewall 14b. The radius of curvature of the seventh sidewall 16a is smaller than that of the eighth sidewall 16b.
[0081] Furthermore, referring to Figure 2 , 9In one embodiment, a removal channel 50 is further provided on the first side of the functional plate 1. The removal channel 50 is connected to the end of the first turning channel 40 away from the fine screening channel 20. The deepening channel 30 extends into the removal channel 50 and is located away from the side where circulating tumor cells 03 accumulate. A diversion hole 51 penetrating the wall of the removal channel 50 is provided in the removal channel 50. Blood continues to flow in the removal channel 50, passing through the diversion hole 51. The circulating tumor cells 03 continue to move along the bottom of the inner wall of the removal channel 50. Since white blood cells 02 and red blood cells 01 are relatively evenly distributed in the channel, some white blood cells 02 and red blood cells 01 flow out from the diversion hole 51, which facilitates the subsequent recovery of circulating tumor cells 03.
[0082] Reference Figure 2 , 8 9. As the liquid flows out of the diversion hole 51, the flow velocity of the liquid in the flow channel will decrease relatively, and the trajectory of the circulating tumor cells 03 will change slightly. They will tend to move away from the inner wall of the flow channel. The circulating tumor cells 03 tend to approach the diversion hole 51. By connecting a first turning flow channel 40 with a large radius of curvature before the removal flow channel 50, the circulating tumor cells 03 will flow close to the bottom of the inner wall of the flow channel after passing through the first turning flow channel 40 with a large bend, thus preventing the circulating tumor cells 03 from flowing into the diversion hole 51 and improving the recovery rate of the circulating tumor cells 03.
[0083] Reference Figure 2 , 3 Furthermore, in one embodiment, a buffer channel 90 with a reciprocating folding structure is provided on the second side of the functional plate 1, and the buffer channel 90 is connected to the diversion hole 51. The buffer channel 90 is designed with a reciprocating folding structure to stabilize the state of the liquid flowing out of the removal channel 50, and to avoid the flow of the remaining liquid in the removal channel 50 being disturbed by shaking, which would affect the subsequent recovery of circulating tumor cells 03.
[0084] The length of the buffer channel 90 controls the amount of liquid discharged. The length of the buffer channel 90, combined with the position of the diversion orifice 51, regulates the amount of leukocytes O2 and erythrocytes O1 discharged and controls the movement trajectory of circulating tumor cells O3. In one embodiment, the proportion of leukocytes O2 and erythrocytes O1 removed by the diversion orifice 51 is 30%-70%, preferably 45%-60%. Similarly, the proportion of liquid flowing out of the diversion orifice 51 is 30%-70%, with an optimal range of 45%-60%. This setting ensures that the liquid outflow is less than 70%, and the final flow rate is reduced by no more than 70%, avoiding interference with the original movement trajectory of the circulating tumor cells O3, preventing some circulating tumor cells O3 from being difficult to recover, and thus reducing the final recovery rate.
[0085] Reference Figure 2 ,9 In one embodiment, a blocking member 52 is provided in the removal channel 50 corresponding to the inlet of the diversion hole 51, and the blocking member 52 is located on the side of the diversion hole 51 away from the deepened channel 30. The width of the blocking member 52 along the extension direction of the removal channel 50 is greater than the diameter of the diversion hole 51. The blocking member 52 protects the circulating tumor cells 03 from flowing into the diversion hole 51. Since the white blood cells 02 and red blood cells 01 are relatively evenly distributed in the channel, the white blood cells 02 and red blood cells 01 flow out from the diversion hole 51 and flow vertically into the second side of the functional plate 1, flowing into the buffer channel 90.
[0086] Furthermore, the blocking member 52 protrudes from the wall of the removal channel 50, meaning that the height of the blocking member 52 is higher than other parts of the removal channel 50, blocking the circulating tumor cells 03 between them and the diversion hole 51, preventing the circulating tumor cells 03 from flowing out of the diversion hole 51. The side of the blocking member 52 near the aggregation of circulating tumor cells 03 is an arc-shaped wall, and the bending direction of the arc-shaped wall is consistent with the bending direction of the side wall of the removal channel 50 near the aggregation of circulating tumor cells 03. The arc-shaped wall of the blocking member 52 near the aggregation of circulating tumor cells 03 matches the aggregation zone of circulating tumor cells 03, ensuring that the effect of the blocking member 52 on the aggregation of circulating tumor cells 03 is consistent and avoiding disruption of the aggregation of circulating tumor cells 03.
[0087] Optionally, the blocking member 52 and the functional plate 1 are integrally formed, and the blocking member 52 is formed by removing the protruding part reserved during the opening of the flow channel 50. The middle part of the side of the blocking member 52 away from the arc-shaped wall is arranged around the outer periphery of the corresponding side of the diversion hole 51. White blood cells O2 and red blood cells O1 flowing towards the diversion hole 51 are blocked by the side wall of the blocking member 52 away from the arc-shaped wall and finally flow into the diversion hole 51.
[0088] Reference Figure 2 , 39. In one embodiment, a plurality of diversion holes 51 are sequentially provided along the extension direction of the removal channel 50, and a plurality of buffer channels 90 with a reciprocating folding structure are provided on the second side of the functional plate 1. The buffer channels 90 are connected to the diversion holes 51 one by one. The multiple diversion holes 51 along the liquid flow direction remove white blood cells O2 and red blood cells O1 multiple times, which is beneficial to the subsequent recovery of circulating tumor cells O3. Since there is no need to aggregate white blood cells O2 and red blood cells O1, the width and height of the channel can also be designed to be wider, which is conducive to increasing the liquid flow rate and improving the sorting efficiency. Meanwhile, a blocking member 52 is provided at the inlet of each corresponding diversion hole 51 in the removal channel 50, and the blocking member 52 is located on the side of the diversion hole 51 away from the deepened channel 30. The width of the blocking member 52 along the extension direction of the removal channel 50 is greater than the diameter of the diversion hole 51. The blocking member 52 protects the circulating tumor cells 03 from flowing into the diversion hole 51, while the white blood cells 02 and red blood cells 01 flow out from the diversion hole 51 and flow vertically into the second side of the functional plate 1 and into the buffer channel 90.
[0089] Reference Figure 9 In one embodiment, along the sample flow direction, the distance L between the plurality of diversion holes 51 and the corresponding deepening channels 30 gradually increases. As the liquid is discharged, leukocytes O2 and erythrocytes O1 gradually flow away from the deepening channels 30. Correspondingly, along the sample flow direction, the diversion holes 51 are gradually positioned away from the deepening channels 30 to facilitate the outflow of leukocytes O2 and erythrocytes O1 from the subsequent diversion holes 51.
[0090] In one embodiment, along the sample flow direction, the length of the buffer channel 90 corresponding to the diversion hole 51 near the first turning channel 40 is greater than the length of the other buffer channels 90. (Refer to...) Figure 3 It can be seen that the length of the buffer channel 90 corresponding to the first diversion hole 51 is longer than that of the others. Since the amount of liquid flowing into the first diversion hole 51 is relatively more than that of the other diversion holes 51, the buffer channel 90 is designed to be longer so that more liquid can flow out, making the outflowing liquid more stable and avoiding affecting the flow rate of subsequent liquids and disrupting the flow of circulating tumor cells 03.
[0091] In one embodiment, the fine screening channel 20 and the removal channel 50 are sinusoidal arc-shaped channels. When the fluid flows in the arc-shaped channel, the parabolic flow of the fluid has the highest velocity in the middle of the channel. When passing through the bend of the channel, the fluid in the middle of the microchannel experiences the greatest centrifugal force due to its maximum velocity, thus flowing towards the outer wall of the arc-shaped channel. The fluid near the channel wall has the lowest velocity and experiences the least centrifugal force, thus being squeezed by the high-velocity fluid in the middle. In order to maintain mass conservation throughout the fluid, a pair of counter-rotating and symmetrical vortices are formed in the direction perpendicular to the fluid flow, located at the upper and lower parts of the channel cross-section, respectively, thereby generating a secondary flow of Dean's vortex. Dean's vortex exerts a drag force on the particles in the fluid, known as Dean's drag. In the arc-shaped channel, the flowing particles are simultaneously subjected to inertial lift and Dean's drag, and the relative magnitudes of these two forces determine the focusing flow of particles in the arc-shaped channel. In this embodiment, due to the effects of inertial lift and Dean's drag in the fine screening channel 20 and the removal channel 50, the circulating tumor cells 03 are focused into a band on the inner wall of the channel.
[0092] Furthermore, referring to Figure 2 , 10 In one embodiment, a second turning channel 60 is further provided on the first side of the functional plate 1. The radius of curvature of the second turning channel 60 is larger than that of the removal channel 50. The second turning channel 60 is connected to the end of the removal channel 50 away from the first turning channel 40. The deepening channel 30 extends into the second turning channel 60 and is located away from the side where circulating tumor cells O3 accumulate. After the blood flows through the diversion orifice 51, the content of white blood cells O2 and red blood cells O1 gradually decreases, and the flow rate also gradually decreases. By connecting the second turning channel 60, whose radius of curvature is larger than that of the removal channel 50, the movement trajectory of the circulating tumor cells O3 can be stabilized, facilitating the subsequent recovery of the circulating tumor cells O3.
[0093] Reference Figure 10 In one embodiment, the second turning channel 60 has an independent recovery channel 70 and a waste liquid channel 80 at its end away from the removal channel 50. The recovery channel 70 is connected to the side of the second turning channel 60 near where circulating tumor cells 03 are concentrated, and the waste liquid channel is connected to the side of the second turning channel 60 near the deepening channel 30. Circulating tumor cells 03 flow into the recovery channel 70 while adhering to the inner wall of the second turning channel 60, while white blood cells 02 and red blood cells 01 flow into the waste liquid channel 80. In one embodiment, the waste liquid channel and the recovery channel 70 have a reciprocating folding structure, which stabilizes the liquid flow and prevents the liquid from falling from the waste liquid hole 32 and the recovery hole 31, thus affecting the movement trajectory of the circulating tumor cells 03 at the end of the second turning channel 60.
[0094] By adjusting the length and depth of the waste liquid channel and the recovery channel 70, the volume ratio of the two can be adjusted, thereby regulating the proportion of leukocyte O2 and erythrocyte O1 removed. In one embodiment, the liquid proportion flowing out of the waste liquid channel is 45%-65%, preferably 50%-60%. The liquid proportion flowing out of the recovery channel 70 is 3%-20%, preferably 5%-10%. The liquid collected from the recovery hole 31 has a circulating tumor cell O3 recovery rate of over 90%, and a leukocyte O2 and erythrocyte O1 removal rate of over 90%. Returning the recovered liquid to the chip for multiple cycles of filtration can further improve the leukocyte O2 and erythrocyte O1 removal rates and increase the purity of the recovered circulating tumor cell O3.
[0095] Reference Figure 1-4 In one embodiment, the upper cover plate 2 is bonded to the first side of the functional plate 1, and the lower cover plate 3 is bonded to the second side of the functional plate 1, so that the flow channels on the first and second sides of the functional plate 1 form a sealed channel. A sample inlet 21 is provided on the upper cover plate 2, which communicates with the initial selection flow channel 10 on the first side of the functional plate 1, so that blood samples can be introduced into the microfluidic chip from the sample inlet 21. The lower cover plate 3 is provided with a recovery hole 31, a waste liquid hole 32, and a discharge hole 33. The diversion hole 51 is connected to the discharge hole 33. Some white blood cells 02 and red blood cells 01 flow through the diversion hole 51 to the second side of the functional plate 1 in the removal channel 50, and flow out of the microfluidic chip from the discharge hole 33 of the lower cover plate 3. The recovery channel 70 is connected to the recovery hole 31 through the first outflow hole 71 that penetrates the first side and the second side of the functional plate. The sorted circulating tumor cells 03 flow out of the microfluidic chip from the recovery hole 31 of the lower cover plate 3. The waste liquid channel is connected to the waste liquid hole 32 through the second outflow hole 81 that penetrates the first side and the second side of the functional plate. The remaining liquid flows out of the microfluidic chip from the waste liquid hole 32 of the lower cover plate 3.
[0096] Traditional microfluidic chips used for the sorting and enrichment of circulating tumor cells (CTCs) are primarily made of PDMS. These chips are expensive, non-reusable, and cost thousands of yuan per test. The fabrication process for PDMS chips limits the design of channels to a single side, restricting their functionality. Furthermore, PDMS chips are not suitable for creating large or deep channels, making it impossible to study multi-level fluid flow states. The small and narrow channels of PDMS also limit the flow rate of the fluid within the chip.
[0097] The microfluidic chip material in the above embodiments is not limited and can be PMMA, PC, ABS, glass, etc. This chip is reusable, requires no antigens or antibodies, and does not require magnetic beads, thus greatly reducing costs. Because this chip only aggregates circulating tumor cells (O3) and not leukocytes (O2) or erythrocytes (O1), the height and width of the flow channels in the chip can be larger, allowing for significant dilution of blood samples, resulting in a higher liquid flow rate and reduced detection time.
[0098] The design strategy of this chip is as follows: circulating tumor cells (03) aggregate into thin bands in the chip's flow channels. By deepening the flow channels (30), leukocytes (02) and erythrocytes (01) are distributed as evenly as possible within the chip, facilitating the recovery of circulating tumor cells (03). The chip design incorporates fundamental principles such as asymmetric inertial focusing and Dean's vortex. A multi-layered design is also employed to facilitate the removal of leukocytes (02). The chip also utilizes a multi-dimensional channel design to reduce the compression of circulating tumor cells (03) within the chip by leukocytes (02).
[0099] Furthermore, the volume of the recovery channel 70 for circulating tumor cells (O3) is very small, while the volume of the waste liquid channel is very large. Therefore, leukocytes (O2) and erythrocytes (O1) can be indirectly removed during the recovery of tumor cells. Because this chip has a high recovery rate for circulating tumor cells (O3), it can be filtered multiple times. Thus, the overall reduction in circulating tumor cells (O3) is relatively small, and leukocytes (O2) are almost completely removed after multiple filtrations.
[0100] One embodiment of this application also provides a method for sorting and enriching circulating tumor cells, which can be implemented using the microfluidic chip for sorting and enriching circulating tumor cells described in any of the above embodiments. The method for sorting and enriching circulating tumor cells in this embodiment includes the following steps:
[0101] Reference Figure 1-4 S100: The diluted blood sample is introduced through the sample inlet 21 of the microfluidic chip. After the blood sample passes through the initial selection channel 10 with a reciprocating folding structure, the circulating tumor cells 03 and white blood cells 02 in the blood sample initially aggregate. The circulating tumor cells 03 aggregate into thin bands and are close to the bottom of the inner wall of the initial selection channel 10, while the white blood cells 02 have not yet aggregated to the bottom of the inner wall of the initial selection channel 10. The red blood cells 01 are dispersed in the initial selection channel 10.
[0102] S200: The blood sample in the initial screening channel 10 is introduced into the fine screening channel 20. A deepened channel 30 is dug on the side of the fine screening channel 20 away from the aggregation of circulating tumor cells 03. The circulating tumor cells 03 in the blood sample aggregate into thin bands and are close to the bottom of the inner wall of the fine screening channel 20, while the white blood cells 02 are in a disordered state and are far away from the bottom of the inner wall of the fine screening channel 20.
[0103] After the blood sample is diluted, it is introduced into the initial screening channel 10 through the sample inlet 21. Due to the influence of inertial lift, Dean's drag, etc., the red blood cells 01 have a smaller diameter and flow chaotically in the initial screening channel 10. The white blood cells 02 and circulating tumor cells 03 have larger diameters and, under the balance of forces in the initial screening channel 10, will initially aggregate into bands and then flow into the fine screening channel 20. The circulating tumor cells 03 have just aggregated into thin bands and are close to the bottom of the inner wall of the fine screening channel 20, while the white blood cells 02 have not yet aggregated to the bottom of the inner wall of the channel. However, the bands of white blood cells 02 and circulating tumor cells 03 are very close together. By excavating a deepening channel 30 on the side of the fine screening channel 20 away from the aggregation of circulating tumor cells 03, the deepening channel 30 flows along the fine screening channel 20. The sieve channel 20 is extended in a certain direction, and the depth of the deepened channel 30 is greater than the depth of the fine sieve channel 20. This disrupts the liquid flow state near the outer wall of the fine sieve channel 20, causing changes in inertial lift and drag force, thus disrupting the original balance. This allows leukocytes O2 to move in a disordered manner, resulting in a more uniform distribution of leukocytes O2 in the fine sieve channel 20. This prevents the aggregation of leukocytes O2 from overlapping with circulating tumor cells O3. Similarly, erythrocytes O1 are also distributed more evenly. This ensures that the aggregation of circulating tumor cells O3 is not interfered with, and also prevents leukocytes O2 from accumulating at the bottom of the inner wall of the fine sieve channel 20. This facilitates the subsequent separation of circulating tumor cells O3 from leukocytes O2 and the recovery of circulating tumor cells O3.
[0104] In one embodiment, the method for sorting and enriching circulating tumor cells further includes the following steps:
[0105] S300: The blood sample in the fine screening channel 20 is introduced into a first turning channel 40 with a radius of curvature larger than that of the fine screening channel 20. Circulating tumor cells O3 in the blood sample aggregate into thin bands and further approach the bottom of the inner wall of the first turning channel 40. (Refer to...) Figure 2 , 8 9. Since the liquid will flow out from the diversion hole 51 in the subsequent S400 step, the flow velocity of the liquid in the channel will be relatively low. The trajectory of the circulating tumor cells 03 will change slightly, and they will tend to move away from the inner wall of the channel. The circulating tumor cells 03 tend to approach the diversion hole 51. By connecting a first turning channel 40 with a large radius of curvature before removing the channel 50, the circulating tumor cells 03 will flow close to the bottom of the inner wall of the channel after passing through the first turning channel 40 with a large bend, thus preventing the circulating tumor cells 03 from flowing into the diversion hole 51 and improving the recovery rate of the circulating tumor cells 03.
[0106] S400: The blood sample in the first turning channel 40 is introduced into the removal channel 50 with a diversion hole 51. Some of the red blood cells O1 and white blood cells O2 in the blood sample flow out of the removal channel 50 through the diversion hole 51, while the circulating tumor cells O3 in the blood sample aggregate into a thin band and approach the bottom of the inner wall of the removal channel 50. The blood continues to flow in the removal channel 50, passing through the diversion hole 51. The circulating tumor cells O3 continue to move along the bottom of the inner wall of the removal channel 50. Because the white blood cells O2 and red blood cells O1 are relatively evenly distributed in the channel, some of the white blood cells O2 and red blood cells O1 flow out from the diversion hole 51, facilitating the subsequent recovery of the circulating tumor cells O3.
[0107] S500: The blood sample remaining after passing through the removal channel 50 is introduced into a second turning channel 60 with a radius of curvature larger than that of the removal channel 50. Circulating tumor cells 03 in the blood sample aggregate into thin bands and further approach the bottom of the inner wall of the second turning channel 60. By connecting the second turning channel 60 with a radius of curvature larger than that of the removal channel 50, the movement trajectory of the circulating tumor cells 03 can be stabilized, facilitating subsequent recovery of the circulating tumor cells 03.
[0108] S600, the second turning channel 60 is connected to a recovery channel 70 on the side near where circulating tumor cells 03 accumulate, and the blood sample on that side is recovered. The other side is connected to a waste liquid channel, and the blood sample on that side is collected. Circulating tumor cells 03 flow into the recovery channel 70 close to the inner wall of the second turning channel 60, while white blood cells 02 and red blood cells 01 flow into the waste liquid channel 80.
[0109] In one embodiment, the method for sorting and enriching circulating tumor cells further includes the following steps:
[0110] S700: The blood sample recovered by the recovery channel 70 is reintroduced into the sample inlet 21 of the microfluidic chip, and the aforementioned steps S100-S600 are repeated. The recovered liquid is circulated and filtered through the aforementioned steps S100-S600, which can further improve the removal rate of white blood cells O2 and red blood cells O1, and improve the recovery purity of circulating tumor cells O3.
[0111] In one embodiment, a blocking member 52 is provided in the removal channel 50 corresponding to the inlet of the diversion hole 51, and the blocking member 52 is located on the side of the diversion hole 51 away from the deepened channel 30. The width of the blocking member 52 along the extension direction of the removal channel 50 is greater than the diameter of the diversion hole 51. The blocking member 52 blocks the circulating tumor cells 03 between the circulating tumor cells 03 and the diversion hole 51, preventing the circulating tumor cells 03 from flowing out of the diversion hole 51.
[0112] Furthermore, the proportion of blood samples flowing out through the diversion orifice 51 is 30%-70%. By controlling the liquid outflow, the liquid outflow is kept below 70%, and the final flow rate is reduced by no more than 70%, thus avoiding affecting the original movement trajectory of circulating tumor cells 03, making it difficult to recover some circulating tumor cells 03, and resulting in a decrease in the final recovery rate.
[0113] Furthermore, the proportion of blood samples flowing out through the waste liquid channel is 45%-65%. By adjusting the length and depth of the waste liquid channel and the recovery channel 70, their volume ratios can be adjusted, thereby regulating the proportion of white blood cells O2 and red blood cells O1 removed. The volume ratio of the recovery channel 70 for recovering circulating tumor cells O3 is very small, while the volume ratio of the waste liquid channel is very large. Therefore, white blood cells O2 and red blood cells O1 can be indirectly removed during the recovery of tumor cells.
[0114] Reference Figure 11 This application also provides an in vitro analytical diagnostic instrument, including a main body and a microfluidic chip for sorting and enriching circulating tumor cells (CTCs) as described in any of the above embodiments. The microfluidic chip for sorting and enriching CTCs is used in conjunction with the main body. The microfluidic chip for sorting and enriching CTCs is a consumable used for sorting and enriching circulating tumor cells (CTCs) O3 in blood. For the same blood sample, the recovered CTCs O3 can be repeatedly filtered to improve the purity of the finally recovered CTCs O3.
[0115] In one embodiment, the main body is provided with a chip mounting position for mounting the microfluidic chip for sorting and enriching circulating tumor cells, a mixing chamber for mixing the sample, diluent and lysis buffer, and a recovery chamber for recovering circulating tumor cells. The mixing chamber is connected to the sample inlet of the microfluidic chip for sorting and enriching circulating tumor cells, and the recovery chamber is connected to the recovery orifice from which circulating tumor cells flow out of the microfluidic chip for sorting and enriching circulating tumor cells.
[0116] In one embodiment, the in vitro analytical diagnostic instrument further includes a power system and a control system. The control system controls the power system to inject the sample, diluent, and lysis buffer into the mixing chamber in a specific ratio, and to pass the mixed liquid in the mixing chamber into the microfluidic chip for sorting and enriching circulating tumor cells at a certain flow rate. The power system can be a pneumatic pump or a syringe pump, etc. The mixing chamber has magnetic stirring or aeration mixing functions. Further, the control system also controls the power system to pre-pass a cleaning solution into the mixing chamber and the microfluidic chip for sorting and enriching circulating tumor cells to clean the connecting pipes, the mixing chamber, and the chip and remove air bubbles. Then, the control system issues a command to control the power system to inject the sample, diluent, and lysis buffer into the mixing chamber in a certain ratio, and the liquid in the mixing chamber is passed into the microfluidic chip for sorting and enriching circulating tumor cells by the power system at a certain flow rate. After the sample mixture passes through the microfluidic chip for sorting and enriching circulating tumor cells, the circulating tumor cells are separated and enter the recovery chamber, and the remaining liquid enters the waste liquid chamber.
[0117] In one embodiment, the in vitro analytical diagnostic instrument further includes a first control valve. The main body is also provided with a cleaning fluid chamber, a sample chamber, a diluent chamber, and a lysis buffer chamber. The cleaning fluid chamber, sample chamber, diluent chamber, and lysis buffer chamber are respectively connected to the power system. One end of the first control valve is connected to the outlet of the cleaning fluid chamber, sample chamber, diluent chamber, and lysis buffer chamber, and the other end is connected to the mixing chamber. The power system drives the sample in the sample chamber, the diluent in the diluent chamber, and the lysis buffer in the lysis buffer chamber to flow towards the mixing chamber in a certain proportion. The first control valve controls the opening and closing of the cleaning fluid chamber, sample chamber, diluent chamber, and lysis buffer chamber with the mixing chamber.
[0118] In one embodiment, the in vitro analytical diagnostic instrument further includes a second control valve, one end of which is connected to the mixing chamber and the other end to the recovery chamber, which is also connected to the power system. For complex samples or samples requiring high purity, the liquid in the recovery chamber can be reintroduced into the mixing chamber via the power system and the second control valve, mixed again with the diluent, and then re-passed through the chip for a second filtration. Similarly, a third and fourth filtration can be performed. This results in highly pure circulating tumor cells.
[0119] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.
[0120] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.
[0121] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.
[0122] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.
[0123] It should be noted that when an element is referred to as being "fixed to" or "set on" another element, it can be directly on the other element or there may be an intervening element. When an element is considered to be "connected to" another element, it can be directly connected to the other element or there may be an intervening element. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and similar expressions used herein are for illustrative purposes only and do not represent the only possible implementation.
[0124] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0125] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A microfluidic chip for sorting and enriching circulating tumor cells, characterized in that, Includes a function board, and the first side of the function board is provided with: The initial selection channel, connected to the sample inlet, is used to initially aggregate circulating tumor cells and leukocytes in the sample. The initial selection channel includes an inlet section, a connecting section, and a sorting section connected in sequence. The end of the inlet section away from the connecting section is connected to the sample inlet. The inlet section has a reciprocating folding structure. The sorting section is an asymmetrical wavy channel in the width direction. The sorting section includes a connecting section. The connecting section includes alternating first bend tube units and second bend tube units. The radius of curvature of the first bend tube unit is greater than the radius of curvature of the second bend tube unit. and The fine screening channel is connected to the primary screening channel. The fine screening channel is an asymmetrical wavy channel with alternating large and small turns. A deepening channel is dug on the side of the fine screening channel away from the accumulation of circulating tumor cells. The deepening channel is arranged along the extension direction of the fine screening channel and the depth of the deepening channel is greater than the depth of the fine screening channel.
2. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 1, characterized in that, The depth of the deepened channel is 50μm-200μm greater than the depth of the fine screening channel.
3. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 2, characterized in that, The depth of the deepened channel is 70μm-120μm greater than the depth of the fine screening channel.
4. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 1, characterized in that, The fine screening channel is designed such that the ratio of the size of circulating tumor cells to the hydraulic diameter is less than or equal to 0.
5.
5. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 4, characterized in that, The fine screening channel is designed such that the ratio of the size of circulating tumor cells to the hydraulic diameter is less than or equal to 0.
07.
6. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 5, characterized in that, The fine screening channel is designed such that the ratio of the size of circulating tumor cells to the hydraulic diameter is 0.045 to 0.
065.
7. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 6, characterized in that, The fine screening channel is designed such that the ratio of the size of circulating tumor cells to the hydraulic diameter is 0.05~0.
06.
8. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 1, characterized in that, The first side of the functional panel is also provided with a first turning channel. The radius of curvature of the first turning channel is greater than that of the fine screening channel. The first turning channel flows with the fine screening channel. The deepening channel extends into the first turning channel and is located on the side away from the accumulation of circulating tumor cells.
9. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 8, characterized in that, The first side of the functional panel is also provided with a removal channel, which is connected to the end of the first turning channel away from the fine screening channel. The deepening channel extends into the removal channel and is located on the side away from the accumulation of circulating tumor cells. A diversion hole penetrating the wall of the removal channel is provided in the removal channel.
10. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 9, characterized in that, The first side of the functional panel is also provided with a second turning channel. The radius of curvature of the second turning channel is greater than that of the removal channel. The second turning channel is connected to the end of the removal channel away from the first turning channel. The deepening channel extends into the second turning channel and is located on the side away from the accumulation of circulating tumor cells.
11. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 10, characterized in that, The second turning channel has an independent recovery channel and a waste liquid channel at the end away from the removal channel. The recovery channel is connected to the side of the second turning channel near the accumulation of circulating tumor cells, and the waste liquid channel is connected to the side of the second turning channel near the deepening channel.
12. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 11, characterized in that, The ratio of the liquid flowing out of the waste liquid channel to the liquid flowing out of the recovery channel is 45%-65%: 3%-20%.
13. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 12, characterized in that, The ratio of the liquid flowing out of the waste liquid channel to the liquid flowing out of the recovery channel is 50%-60%: 5%-10%.
14. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 11, characterized in that, The waste liquid flow channel and the recovery flow channel have a reciprocating folding structure.
15. The microfluidic chip for sorting and enriching circulating tumor cells according to any one of claims 9-14, characterized in that, The second side of the functional board is provided with a buffer channel with a reciprocating folding structure, and the buffer channel is connected to the flow divider hole.
16. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 15, characterized in that, The proportion of liquid flowing out of the buffer channel is 30%-70%.
17. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 16, characterized in that, The proportion of liquid flowing out of the buffer channel is 45%-60%.
18. The microfluidic chip for sorting and enriching circulating tumor cells according to any one of claims 9-14, characterized in that, A blocking element is provided in the removal channel at the inlet of the diversion hole, and the blocking element is located on the side of the diversion hole away from the deepening channel. The width of the blocking element along the extension direction of the removal channel is greater than the diameter of the diversion hole.
19. The microfluidic chip for sorting and enriching circulating tumor cells according to any one of claims 9-14, characterized in that, Multiple diversion holes are sequentially provided along the extension direction of the removal channel. Multiple buffer channels with a reciprocating folding structure are provided on the second side of the functional plate. The buffer channels are connected to the diversion holes one by one. A blocking member is provided in the removal channel at the inlet of the diversion hole. The blocking member is located on the side of the diversion hole away from the deepening channel. The width of the blocking member along the extension direction of the removal channel is greater than the diameter of the diversion hole.
20. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 19, characterized in that, Along the sample flow direction, the distance between the multiple flow dividers and the corresponding deepened channels gradually increases.
21. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 19, characterized in that, Along the sample flow direction, the length of the buffer channel corresponding to the diversion hole near the first turning channel is greater than the length of other buffer channels.
22. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 1, characterized in that, The connecting section includes a first straight pipe section, a first arc-shaped section, a second straight pipe section, a second arc-shaped section, and a third straight pipe section connected in sequence. The first straight pipe section is connected to the end of the inlet section away from the sample inlet, and the third straight pipe section is connected to the sorting section.
23. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 22, characterized in that, The end of the inlet section is arranged in a clockwise direction and connected to the first straight pipe section.
24. The microfluidic chip for sorting and enriching circulating tumor cells according to claim 22, characterized in that, The sorting section further includes a main pipe section. The connecting section is sequentially connected to the main pipe section. The connecting section is connected to the third straight pipe section. The main pipe section is connected to the fine screening channel. The width of the connecting section perpendicular to its extension direction is 'a', and the width of the main pipe section perpendicular to its extension direction is 'b'. <b。 25. The microfluidic chip for sorting and enriching circulating tumor cells according to any one of claims 9-14, characterized in that, The fine screening channel and the removal channel are sinusoidal arc-shaped channels.
26. The microfluidic chip for sorting and enriching circulating tumor cells according to any one of claims 11-14, characterized in that, It also includes an upper cover plate and a lower cover plate. The upper cover plate has a sample inlet and is stacked and connected to the first side of the functional plate. The lower cover plate has a recycling hole, a waste liquid hole and a discharge hole and is stacked and connected to the second side of the functional plate. The recycling channel is connected to the recycling hole, the waste liquid channel is connected to the waste liquid hole, and the diversion hole is connected to the discharge hole.
27. A method for sorting and enriching circulating tumor cells, characterized in that, The method, applied to the microfluidic chip for sorting and enriching circulating tumor cells according to any one of claims 1-26, comprises the following steps: The diluted blood sample is introduced through the sample inlet of the microfluidic chip. After passing through the initial selection channel with a reciprocating folding structure, the circulating tumor cells and white blood cells in the blood sample initially aggregate. The circulating tumor cells aggregate into thin bands and approach the bottom of the inner wall of the initial selection channel, while the white blood cells have not yet aggregated at the bottom of the inner wall of the initial selection channel, and the red blood cells are dispersed in the initial selection channel. The blood sample in the initial screening channel is introduced into the fine screening channel. The circulating tumor cells in the blood sample aggregate into thin bands and approach the bottom of the inner wall of the fine screening channel, while the white blood cells are in a disordered state and away from the bottom of the inner wall of the fine screening channel.
28. The method for sorting and enriching circulating tumor cells according to claim 27, characterized in that, It also includes the following steps: The blood sample in the fine screening channel is introduced into a first turning channel with a radius of curvature greater than that of the fine screening channel. The circulating tumor cells in the blood sample aggregate into thin bands and move closer to the bottom of the inner wall of the first turning channel. The blood sample in the first turning channel is introduced into the removal channel with a diversion hole. The red blood cells and white blood cells in the blood sample flow out of the removal channel through the diversion hole, while the circulating tumor cells in the blood sample aggregate into thin bands and approach the bottom of the inner wall of the removal channel. The blood sample remaining after the removal channel is introduced into a second turning channel with a radius of curvature larger than that of the removal channel. The circulating tumor cells in the blood sample aggregate into thin bands and move closer to the bottom of the inner wall of the second turning channel. The second turning channel is connected to a recovery channel on the side near the accumulation of circulating tumor cells, and the blood sample on that side is recovered. The other side is connected to a waste liquid channel, and the blood sample on that side is collected.
29. The method for sorting and enriching circulating tumor cells according to claim 28, characterized in that, It also includes the following steps: The blood sample recovered from the recovery channel is then reintroduced into the sample inlet of the microfluidic chip, and the aforementioned steps are repeated.
30. The method for sorting and enriching circulating tumor cells according to claim 28 or 29, characterized in that, The removal channel is provided with a blocking member at the inlet of the diversion hole, and the blocking member is located on the side of the diversion hole away from the deepening channel. The width of the blocking member along the extension direction of the removal channel is greater than the diameter of the diversion hole. The proportion of blood samples flowing out through the diversion hole is 30%-70%; the proportion of blood samples flowing out through the waste liquid channel is 45%-65%.
31. An in vitro analytical diagnostic instrument, characterized in that, The invention includes a main body and a microfluidic chip for sorting and enriching circulating tumor cells as described in any one of claims 1-26, wherein the microfluidic chip for sorting and enriching circulating tumor cells can be used in conjunction with the main body.
32. The in vitro analytical diagnostic instrument according to claim 31, characterized in that, The main body is provided with a chip mounting position for mounting the microfluidic chip for sorting and enriching circulating tumor cells, a mixing chamber for mixing the sample, diluent and lysis buffer, and a recovery chamber for recovering circulating tumor cells. The mixing chamber is connected to the sample inlet of the microfluidic chip for sorting and enriching circulating tumor cells, and the recovery chamber is connected to the recovery orifice of the microfluidic chip from which circulating tumor cells flow out.
33. The in vitro analytical diagnostic instrument according to claim 32, characterized in that, It also includes a power system and a control system, wherein the control system is used to control the power system to inject the sample, diluent and lysis buffer into the mixing chamber in a specific ratio and to pass the mixed liquid in the mixing chamber into the microfluidic chip for sorting and enriching circulating tumor cells at a certain flow rate.
34. The in vitro analytical diagnostic instrument according to claim 33, characterized in that, It also includes a first control valve. The main body is further provided with a cleaning fluid chamber, a sample chamber, a diluent chamber, and a lysis fluid chamber. The cleaning fluid chamber, sample chamber, diluent chamber, and lysis fluid chamber are respectively connected to the power system. One end of the first control valve is connected to the outlet of the cleaning fluid chamber, sample chamber, diluent chamber, and lysis fluid chamber, and the other end is connected to the mixing chamber.
35. The in vitro analytical diagnostic instrument according to claim 33 or 34, characterized in that, It also includes a second control valve, one end of which is connected to the mixing chamber and the other end of which is connected to the recovery chamber, which is also connected to the power system.
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