A microfluidic chip for separating CTCs based on size and aptamer

Abstract

This invention belongs to the field of cell sample detection and provides a microfluidic chip for separating circulating tumor cells (CTCs) based on size and aptamers. The invention includes a functional layer with a sample outlet and a sample inlet, and a cell substrate disposed on the upper and lower surfaces of the functional layer. The sample outlet and sample inlet of the functional layer are connected by a separation channel, which includes at least one micropillar array capable of carrying an EpCAM aptamer. The micropillar array is composed of a capture channel surrounding at least two micropillar structures. One end of the micropillar array is connected to the sample inlet, and the other end of the micropillar array has a gradient retention channel connected to the sample outlet. The microfluidic chip uses EpCAM aptamers to modify the micropillar array, increasing the number of collisions between CTCs and the chip, and increasing the probability of collisions between circulating tumor cells and the aptamers on the micropillar surface. Secondly, by utilizing the high specificity of the aptamer for tumor cells, the accuracy and specificity of separation are improved.

Description

Technical Field

[0001] This invention belongs to the field of cell sample detection technology and provides a microfluidic chip for separating CTCs based on size and aptamers. Background Technology

[0002] Circulating tumor cells (CTCs) refer to tumor cells that detach from primary or metastatic tumors and enter the peripheral blood circulation system. Although few in number, they are crucial for early tumor diagnosis, treatment monitoring, and prognostic assessment. In the field of biomedical detection, the isolation and detection of CTCs has always been a key step in cancer research and clinical diagnosis. They are irreplaceable in early cancer diagnosis, precise treatment planning, and prognostic assessment. However, the isolation and detection of CTCs currently faces many serious challenges. Common CTC isolation methods mainly include those based on physical characteristics, such as density, size, and charge, and those based on biological characteristics, such as surface markers. Among physical characteristic-based methods, density gradient centrifugation utilizes differences in cell density to separate cells into layers through centrifugation. However, this method has poor specificity for CTCs, easily confusing them with other blood cells, resulting in low isolation purity. Moreover, this method is difficult to effectively separate smaller CTCs because their density is not significantly different from other blood cells, making precise stratification during centrifugation difficult. While size-based filtration separation methods can perform initial screening based on cell size, traditional filter membrane pore sizes are difficult to precisely adapt to small CTCs, often resulting in incomplete CTC retention or excessive retention of other impurity cells, thus affecting separation efficiency and purity. Immunomagnetic bead separation, based on biological characteristics, utilizes the binding of antibodies on the surface of immunomagnetic beads to specific antigens on the surface of CTCs, achieving CTC separation through a magnetic field. However, this method relies on known tumor cell surface markers, and tumor cells are highly heterogeneous; tumor cell surface markers may differ between different patients, and even within the same patient at different stages, limiting the method's versatility. Furthermore, the binding process of immunomagnetic beads to cells may affect cell viability, hindering subsequent detection and analysis. Summary of the Invention

[0003] The purpose of this invention is to increase the number of collisions between circulating tumor cells (CTCs) and the chip by using a micropillar array, thereby increasing the probability of collisions between circulating tumor cells and aptamers on the micropillar surface, and thus achieving the separation and detection of circulating tumor cells. Secondly, by utilizing the high specificity of aptamers for tumor cell recognition, the accuracy and specificity of separation are improved, overcoming the problem of insufficient specificity in existing technologies.

[0004] A microfluidic chip for separating CTCs based on size and aptamers includes a functional layer with a sample outlet and a sample inlet, and a cell substrate disposed on the upper and lower surfaces of the functional layer. The sample outlet and sample inlet of the functional layer are connected by a separation channel. The separation channel includes at least one micropillar array capable of carrying an EpCAM aptamer. The micropillar array is composed of a capture channel surrounding at least two micropillar structures. One end of the micropillar array is connected to the sample inlet, and the other end of the micropillar array is provided with a gradient trapping channel connected to the sample outlet.

[0005] The microfluidic chip is modified with EpCAM aptamers, which enable the micropillar array surface to specifically recognize and capture circulating tumor cells (CTCs). The above setup extends the length of the capture channel by having it bypass the micropillar array, and allows complex cell samples to repeatedly collide with the micropillar array within the capture channel as it bends, thereby increasing the probability of collisions between CTCs and the aptamers on the micropillar surface, thus achieving the separation and detection of CTCs. As a safety measure, a gradient retention channel is used to retain the remaining complex cell samples after most CTCs have been captured and separated by the micropillar array.

[0006] Preferably, the functional layer uses a rectangular area to set up a micro-pillar array, with the entrance and exit of the micro-pillar array on the same straight line, and the path of the capture channel is a continuous curve arranged in a repeating pattern, with the repeating curve filling the space within the rectangular area.

[0007] Preferably, the microcolumn structure includes a microcolumn and a reagent channel inside the microcolumn. The reagent channel connects the upper surface of the microcolumn and the side surface of the microcolumn. The upper surface of the microcolumn is provided with a reagent inlet of the reagent channel. The inner wall of the capture channel includes the side of the microcolumn that can carry the EpCAM aptamer. The reagent outlet of the reagent channel is the side of the microcolumn that can carry the EpCAM aptamer.

[0008] The surface of the microfluidic chip uses antibiotic protein or streptavidin as a universal binding platform on the micropillar surface. These proteins are covalently immobilized on the micropillar surface via an EDC / NHS cross-linking reaction. Aminated avidin binds to the pre-modified carboxyl groups of the micropillars, utilizing its affinity for biotin to achieve stable binding of the desulfurized biotinylated EpCAM aptamer. A reagent containing a specific single-stranded DNA aptamer for desulfurized biotinylated EpCAM is injected into the micropillar structure through reagent injection ports on the micropillar surface, immobilizing the aptamer on the microfluidic chip's micropillar structure. During subsequent elution, the active ingredient of the eluent competes with the avidin on the micropillar surface for binding, replacing the already bound desulfurized biotinylated aptamer-CTCs complex, allowing both the ligand and CTCs to be eluted together, thus achieving the regeneration and reuse of the microfluidic chip.

[0009] Preferably, the microcolumn structure also includes an elastic seal, with an annular elastic seal at the reagent inlet of the reagent channel. The elastic seal ensures the sealing of the reagent inlet, preventing complex samples from leaking back along the reagent channel from the reagent inlet.

[0010] Preferably, the reagent outlet of the reagent channel is a transverse groove surrounding the side of the micropillar, occupying a central angle α of the micropillar, with a value of 90°≤α≤180°. The reagent outlet surrounding the side of the micropillar allows the reagent containing the desulfurized biotinylated EpCAM-specific single-stranded DNA aptamer to fully wet all directions of the micropillar's side surface, fixing the aptamer onto the micropillar structure of the microfluidic chip and increasing the probability of collision between circulating tumor cells and the aptamers on the micropillar surface.

[0011] Preferably, a capture groove structure capable of carrying EpCAM aptamers is provided below the reagent outlet. The capture groove structure is a vertical groove extending from the lower edge of the reagent outlet to the plane where the cell substrate is located. The above configuration uses a capture groove structure to increase the effective collision area and expand the collision angle range of the micropillar, thereby increasing the probability of circulating tumor cells colliding and being immobilized by the aptamers on the surface of the micropillar. The capture groove structure also has a guiding effect on reagents containing EpCAM aptamers, ensuring that the reagents containing EpCAM aptamers are evenly distributed in all directions of the micropillar structure.

[0012] Preferably, the capture channel structure consists of strip-shaped protrusions spaced at equal angles on the side of the micropillar, with rounded edges along the micropillar axis. The rounded edges of the strip-shaped protrusions along the micropillar axis minimize collision damage to complex cell samples within the capture channel.

[0013] Preferably, the micropillar structure also includes a magnetic microsphere with an EpCAM aptor mounted below the micropillar, a micropillar seat at the bottom of the micropillar that cooperates with the magnetic microsphere, and a gap between the magnetic microsphere and the micropillar seat that serves as a reagent channel.

[0014] Magnetic microspheres rotate within a micropillar-shaped support under the flow impact of complex cell samples. During rotation, each surface of the magnetic microsphere sequentially contacts the reagent containing the EpCAM aptamer, enabling the surface of the magnetic microsphere to be modified with the EpCAM aptamer and bind CTCs. Compared to the cylindrical surface structure of the micropillar structure, the surface structure of the magnetic microsphere increases the contact area with the EpCAM aptamer and CTCs, improving the binding efficiency. After capture, it can be enriched and recycled with the assistance of an external magnetic field, which is beneficial for subsequent cell culture, single-cell sequencing, and microfluidic chip regeneration, reducing chip consumable costs.

[0015] Preferably, the reagent inlet is provided with a reagent groove extending along the axis of the micropillar, and the reagent groove extends from the edge of the reagent inlet to the magnetic microsphere.

[0016] Magnetic microspheres rotate within a micropillar holder under the flow impact of complex cell samples. Reagents containing EpCAM aptamers continuously flow out through the reagent tank to wet the surface of the magnetic microspheres, preventing the magnetic microspheres from getting stuck in the micropillar holder and preventing the reagents from flowing out. This achieves the modification of the magnetic microsphere surface with EpCAM aptamers and the binding of CTCs.

[0017] Preferably, the inlet and outlet heights of the gradient trapping channel are between 15-17 μm, with the inlet height being greater than the outlet height. The diameter of CTCs is between 17-52 μm, larger than that of RBCs (6-8 μm) and WBCs (mostly 7-15 μm). CTCs of various sizes bind to aptamers modified on the chip and are thus trapped, while RBCs and WBCs flow directly out through the channel. Some CTCs that do not bind to aptamers are trapped by the lower-height gradient trapping channel, while RBCs and WBCs can still pass through.

[0018] This invention offers the following advantages: The design of the capture channel, which winds around the micropillar array, extends the channel length, increases the collision probability between CTCs and aptamers, and improves capture efficiency; the gradient retention channel utilizes size differences as a safety measure to effectively retain CTCs not bound by aptamers, achieving precise separation; the combination of reagent channels and elastic seals inside the micropillars ensures uniform modification of the EpCAM aptamer and prevents sample leakage; the capture groove structure increases the collision area and angle while reducing cell damage; the microfluidic chip modifies the capture molecules, and the aptamer is stably immobilized and re-bound to CTCs via reagent injection. During elution, the complex of aptamer and CTCs is eluted, enabling chip regeneration and reducing consumable costs. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the functional layer and cell substrate of the present invention.

[0021] Figure 2 This is a schematic diagram of a microfluidic chip based on EpCAM aptamer-separated CTCs.

[0022] Figure 3 This is a front view of the functional layer of the present invention.

[0023] Figure 4The functional layer and cell substrate of the present invention Figure 1 sectional view

[0024] Figure 5 This is a rear view of the functional layer of the present invention.

[0025] Figure 6 This is a schematic diagram of the micropillar structure according to Embodiment 1 of the present invention.

[0026] Figure 7 This is a top view of the micropillar structure according to Embodiment 1 of the present invention.

[0027] Figure 8 This is a front view of the micropillar structure according to Embodiment 1 of the present invention.

[0028] Figure 9 The micropillar structure of Embodiment 1 of the present invention Figure 8 A sectional view.

[0029] Figure 10 This is a schematic diagram of the micropillar structure of Embodiment 2 of the present invention.

[0030] Figure 11 This is a schematic diagram of the micropillar structure of Embodiment 2 of the present invention.

[0031] Legend: 1 Functional layer; 2 Cell substrate; 11 Sample inlet; 12 Micropillar array; 121 Micropillar structure; 121a Elastic seal; 121b Reagent channel; 121c Capture groove structure; 121d Reagent groove; 121e Magnetic microspheres; 121f Micropillar ball seat; 122 Capture channel; 13 Gradient retention channel; 14 Sample outlet. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0033] like Figure 1 , Figure 2 and Figure 5 As shown, a microfluidic chip for separating CTCs based on size and aptamers includes a functional layer 1 with a sample outlet 14 and a sample inlet 11, and a cell substrate 2 disposed on the upper and lower surfaces of the functional layer 1. The sample outlet 14 and the sample inlet 11 of the functional layer 1 are connected by a separation channel. The separation channel includes two symmetrical micropillar arrays 12 capable of carrying EpCAM aptamers. The micropillar arrays 12 are composed of multiple micropillar structures 121 around which the capture channel 122 is arranged. One end of the micropillar array 12 is connected to the sample inlet 11, and the other end of the micropillar array 12 is provided with a gradient trapping channel 13 connected to the sample outlet 14.

[0034] The microfluidic chip is modified with EpCAM aptamers, giving the surface of the micropillar array 12 EpCAM aptamers for specific recognition and capture of circulating tumor cells (CTCs). The above setup extends the length of the capture channel 122 by winding around the micropillar array 12, and causes complex cell samples to repeatedly collide with the micropillar array 122 within the capture channel 122 as it bends repeatedly, thereby increasing the probability of collision between CTCs and the aptamers on the micropillar surface, thus achieving the separation and detection of CTCs. The gradient interception channel 13 serves as a safety measure. After most CTCs are captured and separated by the micropillar array 12, CTCs in the remaining complex cell samples that have not bound aptamers or whose bound aptamers have accidentally detached are intercepted due to exceeding the size limit, while blood cells pass through smoothly. This forms a dual guarantee of specific recognition and size screening, reducing the false negative rate.

[0035] like Figure 2 and Figure 3 As shown, functional layer 1 uses a rectangular area to set up a micropillar array 12. The inlet and outlet of the micropillar array 12 are on the same straight line. The path of the capture channel 122 is a continuous curve arranged in a repetitive pattern, and the repetitive curve fills the space within the rectangular area. Setting the inlet and outlet on the same straight line in the rectangular area reduces backflow and dead zones; the continuous curve channel extends the channel length within a limited space, and the repeated collisions and deceleration of the curve channel make the sample flow rate uniform, avoiding local high flow rates that could lead to missed detection of CTCs; at the same time, the repetitive and regular curve layout facilitates automated fluorescence microscopy to scan line by line, shortening the detection time and improving the efficiency of batch sample detection.

[0036] like Figure 4 and Figure 5 As shown, the inlet and outlet heights of the gradient retention channel 13 are between 15-17 μm, with the inlet height being greater than the outlet height. The diameter of CTCs, between 17-52 μm, is larger than that of RBCs (6-8 μm) and WBCs (7-15 μm). CTCs of all sizes bind to the aptamers modified on the chip and are thus captured, while RBCs and WBCs flow directly out through the channel. Some CTCs that do not bind to the aptamers are retained by the lower-height gradient retention channel 13, while RBCs and WBCs can still pass through. After immunofluorescence staining, the CTCs captured on the chip show DAPI (4',6-diamidinyl-2-phenylindole) positivity, EpCAM positivity, and CD45 negativity under a fluorescence microscope; while the retained WBCs of the same size show DAPI positivity, EpCAM negativity, and CD45 positivity after immunofluorescence staining.

[0037] Example 1

[0038] refer to Figure 3 and Figure 6 As shown, the micropillar structure 121 includes a micropillar and a reagent channel 121b inside the micropillar. The reagent channel 121b connects the upper surface of the micropillar and the side surface of the micropillar. The upper surface of the micropillar is provided with a reagent inlet of the reagent channel 121b. The inner wall of the capture channel 122 includes the side surface of the micropillar that can carry the EpCAM aptamer. The reagent outlet of the reagent channel 121b is the side surface of the micropillar that can carry the EpCAM aptamer.

[0039] The surface of the microfluidic chip utilizes antibiotic protein or streptavidin as a universal binding platform on the micropillar surface. These proteins are covalently immobilized on the micropillar surface via an EDC / NHS cross-linking reaction. Aminated avidin binds to the pre-modified carboxyl groups of the micropillars, leveraging its affinity for biotin to achieve stable binding of the desulfurized biotinylated EpCAM aptamer. A reagent containing the desulfurized biotinylated EpCAM-specific single-stranded DNA aptamer is injected into the micropillar structure 121 through a reagent injection port on the micropillar surface, immobilizing the aptamer on the microfluidic chip's micropillar structure 121. During subsequent elution, the free biotin in the eluent competes with the avidin on the micropillar surface for binding, replacing the already bound desulfurized biotinylated ligand-CTCs complex, allowing both the ligand and CTCs to be eluted together, or at pH 4.0. The microfluidic chip can be regenerated and reused by gentle dissociation in an acidic buffer solution. It can selectively inject desulfurized biotinylated aptamer reagents, and is not limited to using EpCAM aptamer to bind with CTCs alone. It can be extended to other reagents to capture tumor cells or other cells.

[0040] Example 2

[0041] Based on Example 1, such as Figure 6 , Figure 7 and Figure 9 As shown, the micropillar structure 121 also includes an elastic seal 121a, and the reagent inlet of the reagent channel 121b is provided with an annular elastic seal 121a. The elastic seal 121a ensures the sealing of the reagent inlet and prevents complex samples from leaking back along the reagent channel 121b from the reagent inlet.

[0042] like Figure 7 and Figure 8 As shown, the reagent outlet of reagent channel 121b is a transverse groove surrounding the side of the micropillar, occupying the central angle α of the micropillar, with a value of 90°≤α≤180°. The reagent outlet surrounding the side of the micropillar allows the reagent containing the desulfurized biotinylated EpCAM-specific single-stranded DNA aptamer to fully wet all directions of the side of the micropillar, fixing the aptamer onto the micropillar structure 121 of the microfluidic chip and increasing the probability of collision between circulating tumor cells and the aptamer on the micropillar surface.

[0043] like Figure 6 As shown, a capture groove structure 121c capable of carrying EpCAM aptamers is provided below the reagent outlet. The capture groove structure 121c is a vertical groove extending from the lower edge of the reagent outlet to the plane where the cell substrate 2 is located. The above-mentioned configuration uses the capture groove structure 121c to increase the effective collision area and expand the collision angle range of the side of the micropillar, thereby increasing the probability of collision fixation between circulating tumor cells and aptamers on the surface of the micropillar. The capture groove structure 121c has a guiding effect on reagents containing EpCAM aptamers, ensuring that the reagents containing EpCAM aptamers are evenly distributed in all directions of the micropillar structure 121. The capture groove serves as a temporary storage area, which can centrally recover CTCs during elution, reduce the shear force on the EpCAM aptamer-CTCs complex after binding, prevent the accidental detachment of the EpCAM aptamer-CTCs complex, and protect the nucleic acid integrity of CTCs, thereby improving the accuracy of gene detection.

[0044] The capture channel structure 121c consists of strip-shaped protrusions spaced at equal angles on the side of the micropillar, with rounded edges along the micropillar axis. The rounded edges of the strip-shaped protrusions along the micropillar axis minimize collision damage to complex cell samples within the capture channel 122. The strip-shaped protrusions and the transverse groove at the reagent outlet easily form micro-vortices, reducing the flow rate of complex cell samples, extending the residence time of CTCs, and improving capture efficiency.

[0045] Example 3

[0046] like Figure 10 and Figure 11 As shown, the difference from Embodiments 1 and 2 is that in this embodiment, the micropillar structure 121 further includes a magnetic microsphere 121e capable of housing the EpCAM aptamer below the micropillar. The bottom of the micropillar is a micropillar seat 121f that cooperates with the magnetic microsphere 121e. A gap between the magnetic microsphere 121e and the micropillar seat 121f serves as a reagent channel 121b. Under the flow impact of complex cell samples, the magnetic microsphere 121e rotates within the micropillar seat 121f. During the rotation, each surface of the magnetic microsphere 121e sequentially contacts the reagent containing the EpCAM aptamer, achieving surface modification of the magnetic microsphere 121e with the EpCAM aptamer and binding of CTCs. Compared to the cylindrical surface structure of the micropillar structure 121, the surface structure of the magnetic microsphere 121e increases the contact area with the EpCAM aptamer and CTCs, improving the binding efficiency. During the capture and elution process, an external magnetic field can be used to assist in enrichment and recycling, which is beneficial for the subsequent regeneration and directional collection of CTCs in the microfluidic chip, reducing the cost of chip consumables.

[0047] A reagent reservoir 121d extending along the axis of the micropillar is provided at the reagent inlet. The reagent reservoir 121d extends from the edge of the reagent inlet to the magnetic microsphere 121e. The reagent channel 121b between the magnetic microsphere 121e and the micropillar seat 121f is small. Due to factors such as intermolecular forces, the reagent cannot flow out naturally from the reagent channel 121b. Therefore, reagent will remain from the reagent inlet to the top of the magnetic microsphere 121e after the reagent is injected.

[0048] When the magnetic microspheres 121e rotate within the micropillar seat 121f under the flow impact of complex cell samples, the reagent containing the EpCAM aptamer continuously wets the surface of the magnetic microspheres 121e through the reagent tank 121d, preventing the magnetic microspheres 121e and the micropillar seat 121f from getting stuck and the reagent from flowing out, thus achieving the modification of the surface of the magnetic microspheres 121e with the EpCAM aptamer and the binding of CTCs.

[0049] The present invention has the following beneficial effects: the design of the capture channel 122 around the micropillar array 12 extends the channel length, increases the collision probability between CTCs and aptamers, and improves the capture efficiency; the gradient retention channel 13 uses size differences as a safety measure to effectively retain CTCs that have not been bound by the aptamer, achieving precise separation; the combination of the reagent channel 121b and the elastic seal 121a inside the micropillar ensures uniform modification of the EpCAM aptamer and avoids sample leakage; the capture groove structure 121c increases the collision area and angle while reducing cell damage; the microfluidic chip modifies the capture molecules, and the aptamer is stably fixed and re-bound to CTCs by injecting reagents. During elution, the complex of the aptamer and CTCs is eluted to achieve chip regeneration and reduce consumable costs.

[0050] The above embodiments and / or implementation methods are merely illustrative of preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art may make some modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but these should still be regarded as the technology or embodiments that are substantially the same as the present invention.

Claims

1. A microfluidic chip for separating CTCs based on size and aptamers, comprising a functional layer (1) having a sample outlet (14) and a sample inlet (11) and a cell substrate (2) disposed on the upper and lower surfaces of the functional layer (1), characterized in that, The sample outlet (14) and sample inlet (11) of the functional layer (1) are connected by a separation channel. The separation channel includes at least one micropillar array (12) capable of carrying an EpCAM adapter. The micropillar array (12) is composed of a capture channel (122) that surrounds at least two micropillar structures (121). One end of the micropillar array (12) is connected to the sample inlet (11), and the other end of the micropillar array (12) is provided with a gradient retention channel (13) connected to the sample outlet (14). The functional layer (1) uses a rectangular area to set up a micropillar array (12). The entrance and exit of the micropillar array (12) are on the same straight line. The path of the capture channel (122) is a continuous curve arranged in a repeating pattern. The repeating curve fills the space within the rectangular area. Complex cell samples repeatedly collide with the micropillar array (122) in the capture channel (122) as the capture channel (122) bends repeatedly, thereby increasing the collision probability of circulating tumor cells with the EpCAM aptamer on the surface of the micropillar. The micropillar structure (121) includes a micropillar and a reagent channel (121b) inside the micropillar. The reagent channel (121b) connects the upper surface of the micropillar and the side surface of the micropillar. The upper surface of the micropillar is provided with a reagent inlet of the reagent channel (121b). The inner wall of the capture channel (122) includes the side surface of the micropillar that can carry the EpCAM aptamer. The reagent outlet of the reagent channel (121b) is the side surface of the micropillar that can carry the EpCAM aptamer. The micropillar structure (121) also includes an EpCAM aptamer magnetic microsphere (121e) that can be mounted below the micropillar. The bottom of the micropillar is a micropillar seat (121f) that cooperates with the magnetic microsphere (121e). There is a gap between the magnetic microsphere (121e) and the micropillar seat (121f) that serves as a reagent channel (121b).

2. A microfluidic chip for separating CTCs based on size and aptamer according to claim 1, characterized in that, The microcolumn structure (121) also includes an elastic seal (121a), and the reagent inlet of the reagent channel (121b) is provided with an annular elastic seal (121a).

3. A microfluidic chip for separating CTCs based on size and aptamer according to claim 2, characterized in that, The reagent outlet of the reagent channel (121b) is a transverse groove surrounding the side of the micropillar, occupying the central angle α of the micropillar, with a value of 90°≤α≤180°.

4. A microfluidic chip for separating CTCs based on size and aptamer according to claim 3, characterized in that, Below the reagent outlet is a capture groove structure (121c) capable of carrying the EpCAM aptamer. The capture groove structure (121c) is a vertical groove extending from the lower edge of the reagent outlet to the plane where the cell substrate (2) is located.

5. A microfluidic chip for separating CTCs based on size and aptamer according to claim 4, characterized in that, The capture groove structure (121c) is composed of strip-shaped protrusions that are equally spaced on the side of the micropillar, and the edges of the strip-shaped protrusions along the axis of the micropillar are rounded.

6. A microfluidic chip for separating CTCs based on size and aptamer according to claim 1, characterized in that, The reagent inlet is provided with a reagent reservoir (121d) extending along the axis of the microcolumn, and the edge of the reagent inlet in the reagent reservoir (121d) extends to the magnetic microsphere (121e).

7. A microfluidic chip for separating CTCs based on size and aptamer according to claim 1, characterized in that, The inlet height and outlet height of the gradient retention channel (13) are between 15-17 μm, and the inlet height of the gradient retention channel (13) is greater than the outlet height.

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