Microfluidic device for sorting circulating tumor cells and enriching extracellular vesicles

Abstract

The application provides a microfluidic device for sorting circulating tumor cells and enriching exosomes. It comprises a fixed plate, a first sorting module for separating cells and exosomes, an enrichment module for enriching exosomes, and a second sorting module for sorting normal blood cells and circulating tumor cells. The inlet of the enrichment module is connected to the exosome outlet of the first sorting module, and the inlet of the second sorting module is connected to the cell outlet of the first sorting module. The inlet of the sorting module is also provided with a sheath liquid input module for inputting sheath liquid to the second sorting module. The microfluidic device can separate cells and exosomes by using synergistic Dean flow microfluidic inertial sorting, then realize efficient enrichment of exosomes by using a pit-assisted thermal field, and separate tumor cells and normal cells by using deterministic lateral displacement technology (DLD) combined with insulating dielectrophoresis technology (i DEP).

Description

Technical Field

[0001] This invention belongs to the field of microfluidics technology and relates to a microfluidic device that can separate circulating tumor cells and enriched exosomes from a single blood sample. Background Technology

[0002] Circulating tumor cells (CTCs) refer to tumor cells that enter the peripheral blood of the human body. Monitoring the trends in the type and number of CTCs is of great significance for patient prognosis, efficacy evaluation, and personalized treatment. Traditional detection methods use flow cytometry to sort and count cells in blood samples. This method requires expensive laboratory equipment, complex experimental procedures, and consumes a large amount of reagents. Furthermore, the detection process is time-consuming and costly.

[0003] Exosomes are small vesicles containing complex RNA and proteins. Cancer markers (exosomes or exovesicles) are present in the blood in the early stages of cancer, and early diagnosis of cancer can be achieved by detecting these markers. However, the content of exosomes and vesicles in the blood of early-stage cancer patients is extremely low, and the presence of micron-sized cells such as white blood cells and red blood cells results in low sensitivity and accuracy of instruments for detecting exosomes, greatly increasing the difficulty of direct detection.

[0004] In recent years, the rapid development of microfluidic technology has enabled the rapid and precise control of microfluidics or micro-nano particles using microfluidic chips, realizing many functions of traditional laboratories such as separation and detection. It has also brought about tremendous changes in detection technology by having advantages such as high integration, fast processing speed, low reagent consumption, and low cost, making integrated rapid detection instruments possible.

[0005] In microfluidics, particle manipulation methods are categorized into passive and active methods based on the presence or absence of energy input. Passive methods primarily rely on differences in particle size, shape, and density, as well as the inertial and viscous forces of the fluid, to manipulate particles within specific microchannel structures. Active methods utilize external fields (physical fields such as magnetic, acoustic, electric, and thermal fields) to manipulate particles in solution. Magnetic field manipulation requires modification with magnetic beads containing specific antibodies to effectively improve manipulation precision. The particle preparation process is complex, and cleaning the magnetic material after manipulation is difficult, affecting sample performance. Acoustic field manipulation uses ultrasonic fields to induce acoustic flow and apply acoustic radiation forces to particles, causing them to migrate to specific locations. This method allows for simple chip fabrication but results in large sizes, making miniaturization difficult. Optical field manipulation uses focused lasers to apply scattering and gradient forces to particles for manipulation, requiring complex and costly platform equipment.

[0006] Thermophoretic manipulation of microparticles can be divided into static and fluid types. Traditional thermophoretic manipulation is generally static and has relatively low efficiency. Fluid thermophoresis, on the other hand, can combine thermal convection effects to make thermal field manipulation of microparticles more efficient. However, conventional fluid thermophoretic enrichment often has unreasonable structural design, cannot make good use of the synergistic effect of thermophoresis and thermal convection, and has the disadvantage of difficulty in collecting well enriched microparticles.

[0007] Dielectrophoresis manipulation techniques allow for continuous and precise control of unlabeled cells during flow. These techniques can be categorized into conventional dielectrophoresis, insulator-induced dielectrophoresis (iDEP), and contactless dielectrophoresis (cDEP). Conventional and contactless dielectrophoresis require the fabrication of 3D electrodes, resulting in complex chip structures. Furthermore, traditional insulator-induced dielectrophoresis generally utilizes only electroosmotic flow as the driving force, leading to low throughput.

[0008] Furthermore, circulating tumor cells (CTCs) and exosomes are both important biomarkers for early cancer diagnosis. Traditional microfluidic processing methods often focus on enriching only one of them, while treating the other cellular biomarker as waste, which significantly reduces the accuracy of early cancer diagnosis. Therefore, there is an urgent need for a microfluidic solution that can sort CTCs and enrich exosomes from a single blood sample. Summary of the Invention

[0009] The purpose of this invention is to address the aforementioned problems in existing technologies by proposing a low-cost, high-efficiency microfluidic device for sorting circulating tumor cells and enriching exosomes.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] A microfluidic device for separating circulating tumor cells and enriching exosomes includes a fixed plate. The fixed plate is provided with a first sorting module for separating cells and exosomes, an enrichment module for enriching exosomes, and a second sorting module for separating normal blood cells and circulating tumor cells. The inlet of the enrichment module is connected to the exosome outlet of the first sorting module, and the inlet of the second sorting module is connected to the cell outlet of the first sorting module. The inlet of the sorting module is also provided with a sheath fluid input module for inputting sheath fluid into the second sorting module.

[0012] A single whole blood sample to be tested is treated with erythrocyte lysis buffer, centrifuged, and the supernatant is collected. A pressure pump is used to pump the supernatant through the inlet of the first sorting module, where cells and exosomes are sorted to different outlets. Cells enter the second sorting module, where normal blood cells and circulating tumor cells are separated; exosomes enter and accumulate in the enrichment module.

[0013] In the aforementioned microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes, the enrichment module includes a first channel horizontally disposed within a fixed plate, an enrichment cavity disposed at the bottom of the first channel with a circular cross-section, and a laser located directly above the enrichment cavity for heating the suspension within the enrichment cavity. The heating area of ​​the laser is cylindrical and centered with the enrichment cavity, and the outer diameter of the heating area is smaller than the outer diameter of the enrichment cavity. The bottom of the enrichment cavity is provided with a structure for enhancing the synergistic effect of thermophoresis and thermal convection.

[0014] The outer diameter of the enrichment cavity is greater than or equal to the width of the first channel, and the fixing plate is made of a transparent material, such as biocompatible PDMS silicone or plexiglass, to facilitate the transmission of laser light into the enrichment cavity.

[0015] The laser emitted from the laser beam passes through a transparent fixed plate from top to bottom and irradiates the heating area to rapidly and precisely heat the suspension, forming a temperature gradient field that causes the cell exosomes in the suspension to migrate towards the center of the enrichment cavity.

[0016] In the aforementioned microfluidic device capable of sorting circulating tumor cells and enriching exosomes, the structure used to enhance the synergistic effect of thermophoresis and thermal convection is a top cone located at the bottom of the enrichment chamber. The top cone is centered within the enrichment chamber, and its generatrix is ​​an arc. The outer diameter of the top cone gradually decreases from bottom to top. The intersection of the longitudinal section passing through the centerline of the top cone and the outer surface of the top cone forms the generatrix of the top cone; that is, the outer surface of the top cone is an arc surface. This enhances the synergistic effect of thermophoresis and thermal convection, thereby increasing the enrichment efficiency and enrichment factor.

[0017] The apical cone can increase the temperature gradient within the enrichment cavity, enhancing the thermophoresis effect and better utilizing the synergistic effect of thermophoresis and thermal convection to enrich cell exosomes within the enrichment cavity more quickly and efficiently.

[0018] Furthermore, the outer surface of the apex cone is an elliptical arc surface with the concave side facing upwards.

[0019] In the aforementioned microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes, the bottom of the first channel has a circular hole through which a fixing plate is disposed downwards, and an enrichment seat is sealed and fitted inside the circular hole, with the enrichment cavity centered on the enrichment seat.

[0020] By setting a through hole at the bottom of the first channel, the enrichment seat can be detachably placed inside the hole, making it easy to remove the enrichment seat and thus facilitating the collection of cell exosomes.

[0021] In the aforementioned microfluidic device capable of sorting circulating tumor cells and enriching exosomes, a base plate is provided below the fixing plate, a limiting boss is provided on the base plate, the enrichment seat is provided on the limiting boss, a limiting cavity is provided on the lower side of the fixing plate to cooperate with the limiting boss, and a circular hole is provided in the limiting cavity.

[0022] The enrichment seat and the circular through-hole are detachably sealed, facilitating the removal of enriched exosomes for subsequent analysis. The substrate is attached to the lower side of the fixing plate, and a limiting boss engages with the limiting cavity to position the enrichment seat. When the limiting boss engages with the limiting cavity, the enrichment seat fits within the circular hole.

[0023] To improve sealing, an annular gasket is installed between the enrichment seat and the fixing plate.

[0024] In the aforementioned microfluidic device capable of sorting circulating tumor cells and enriched exosomes, the first sorting module includes a curved microchannel disposed within a fixed plate and a rectangular cavity disposed on the inner side of the curved microchannel. The rectangular cavity is a plurality of such cavities and is uniformly arranged along the length direction of the curved microchannel. The plurality of rectangular cavities are connected to the curved microchannel. The cell outlet of the first sorting module is connected to the inner side of the curved microchannel, and the exosome outlet of the first sorting module is connected to the outer side of the curved microchannel.

[0025] The curved microchannel extends horizontally. Under the synergistic effect of the Dean's secondary flow induced by the curved microchannel and the Dean's flow caused by several rectangular cavity arrays, micron-sized cells (blood cells and circulating tumor cells) are single-line focused at almost the same focal center on the curved inner side near the microchannel. Nano-sized exosomes, due to their small particle size, are almost unaffected by the Dean's flow and are uniformly dispersed in the channel, thus sorting cells and exosomes to different outlets.

[0026] In the aforementioned microfluidic device capable of separating circulating tumor cells and enriched exosomes, the second sorting module includes a sorting cavity disposed within a fixed plate, a second channel communicating with the sorting cavity, a normal blood cell outlet, and a circulating tumor cell outlet. The inlet of the second channel is connected to the cell outlet of the first sorting module. A plurality of arrayed micropillars are erected within the sorting cavity. The plurality of micropillars are arranged obliquely along the flow direction of the sheath fluid in the second channel. The normal blood cell outlet is located at the end of the normal blood cell movement direction, and the circulating tumor cell outlet is located at the end of the circulating tumor cell movement direction.

[0027] The second sorting module utilizes deterministic lateral displacement (DLD) technology to separate normal blood cells from circulating tumor cells. DLD is a sorting technique based on particle size and shape. Its principle involves arranging micropillars at a specific angle along the flow direction. Different critical diameters are generated depending on parameters such as the shape and spacing of the micropillars. This causes circulating tumor cells with a diameter larger than the critical diameter and a small number of normal blood cells to move towards the circulating tumor cell outlet, while normal blood cells with a diameter smaller than the critical diameter move to the normal blood cell outlet and are discharged there. The micropillars are insulated.

[0028] In the aforementioned microfluidic device capable of separating circulating tumor cells and enriched exosomes, the sorting chamber is provided with two opposing electrophoresis plates, and a flow channel is formed between the two electrophoresis plates. There are two outlets for circulating tumor cells, which are respectively located close to the inner side of different electrophoresis plates. A second outlet for normal blood cells is provided between the two outlets for circulating tumor cells. The microfluidic device also includes an AC power supply, the negative terminal of which is connected to the second outlet for normal blood cells, and the positive terminal of which is connected to the second channel.

[0029] The second sorting module uses deterministic lateral displacement (DLD) technology in conjunction with insulator-induced dielectrophoresis (iDEP) to separate normal blood cells from circulating tumor cells.

[0030] In the aforementioned microfluidic device capable of sorting circulating tumor cells and enriching exosomes, the sheath fluid input module includes several sheath fluid channels communicating with the second channel, and the positive terminal of the AC power supply is connected to one of the sheath fluid channels.

[0031] The working process of this microfluidic device is as follows:

[0032] A single whole blood sample to be tested is treated with red blood cell lysis buffer, centrifuged, and the supernatant is collected. The supernatant is pumped into the first sorting module through the inlet of the first sorting module using a pressure pump. The sample solution passes through a curved microchannel with a rectangular expansion and contraction array. The Dean's secondary flow induced by the curved microchannel and the Dean's flow caused by several rectangular cavity arrays work together to achieve single-line focusing of micron-sized cells at almost the same focusing center and sort the cells to the cell outlet. Nano-sized exosomes, due to their small particle size, are almost unaffected by the Dean's flow and are uniformly dispersed in the channel and sorted to the exosome outlet.

[0033] The cell solution enters the second sorting module, where it forms a focused flow of three-dimensional monodisperse particles under the action of the sheath fluid. These particles sequentially enter the microcolumn array region. In this region, smaller cells (normal blood cells) flow in a zigzag pattern and migrate laterally to the normal blood cell outlet. Larger blood cells and circulating tumor cells will undergo a collision-like flow under the action of deterministic lateral displacement, gradually migrating towards the circulating tumor cell outlet along the direction of the array's staggered gradient.

[0034] During the migration towards the exit of circulating tumor cells, the non-uniform electric field formed between the insulating micropillars causes the cells to be subjected to a dielectrophoretic force. This dielectrophoretic force reduces the critical diameter for deterministic lateral displacement. Therefore, we can increase the gap between the micropillars to prevent cells from clogging the micropillar array.

[0035] Between the two electrophoresis plates, due to the difference in dielectric properties, normal blood cells are subjected to negative dielectric force, while circulating tumor cells are subjected to positive dielectric force, causing the circulating tumor cells to migrate to the circulating tumor cell outlets on both sides and be collected.

[0036] After the exosomes enter the enrichment module, the cylindrical enrichment cavity in the first channel is gradually filled with the suspension as the exosome suspension is injected. As the suspension containing exosomes is continuously injected, the exosomes in the enrichment cavity gradually migrate towards the pit at the bottom center of the cavity under the combined action of thermophoresis and thermal convection.

[0037] An air inlet is also provided at the entrance of the curved microchannel. Either the entrance of the curved microchannel or the air inlet is opened. After all the supernatant is injected, a certain amount of gas is injected through the air inlet at the same flow rate until the cell solution in the fixation plate is completely discharged.

[0038] After enrichment, the enrichment seat is separated from the well, and the enriched cell exosomes are collected from the enrichment cavity for subsequent biomedical testing (PCR, immunochemical analysis, enzyme analysis, etc.).

[0039] Compared with the prior art, the present invention has the following advantages:

[0040] 1. In the first sorting module, an expansion-contraction array composed of several rectangular cavities is set on the inside of the bend of the microchannel, so that the Dean secondary flow induced by the two structures of the bend microchannel and the rectangular cavity is in the same direction, which synergistically enhances the focusing of microparticles, enabling micron-sized cells to achieve single-line focusing, and the focusing center position of micron-sized cells of different sizes is the same, so as to achieve the purpose of completely separating micron-sized cells, avoiding the mutual influence between micron-sized cells and nano-sized cell exosomes, reducing the required focusing channel length, accelerating the solution processing speed, and improving the detection rate of circulating tumor cells.

[0041] 2. The pit-assisted thermal field manipulation method, compared to other active manipulation methods, eliminates the need for particle labeling, requires less energy, and does not harm biological particles. Compared to traditional methods that use mechanical heating to create a thermal field, resulting in complex chip structures and difficult processing, this invention uses laser heating technology to rapidly and precisely heat the enrichment cavity, forming a constant temperature gradient field, thereby effectively manipulating cell derivatives. Utilizing a special elliptical apical cone structure, it better leverages the synergistic effect of thermophoresis and thermal convection, increasing the temperature gradient at the center of the enrichment cavity, enhancing the thermophoresis effect, and enriching cell exosomes faster and more efficiently. The separable design solves the problem of difficulty in collecting well-enriched particles in traditional flow-through thermophoresis enrichment.

[0042] 3. The second sorting area utilizes the synergistic effect of micropillar arrays and insulating dielectric electrophoresis. Within the micropillar array, deterministic lateral displacement is used to sort cells of different diameters. During the migration process of deterministic lateral displacement, the non-uniform electric field formed between the micropillar arrays allows the distance between the micropillar arrays to be increased without changing the critical diameter, solving the problem of blockage that is common in traditional micropillar arrays. Furthermore, by utilizing the difference in dielectric properties, circulating tumor cells with particle sizes not much different from normal blood cells are migrated towards the circulating tumor cell exit direction, greatly improving the detection rate of circulating tumor cells. Moreover, by using electrodes connected at the second channel instead of traditional embedded three-dimensional electrodes, this sorting method has the advantages of high sorting efficiency, simple structure, and intact biological activity of the sorted circulating tumor cells.

[0043] 4. Compared with traditional methods, this device can simultaneously sort tumor cells and enrich exosomes. Furthermore, it separates the two in advance through microfluidic combined with Dean flow inertial sorting, avoiding cross-influence between the two on subsequent biomedical detection. This greatly improves the early detection rate of cancer and provides a simple, low-cost, and accurate microfluidic manipulation device for detecting circulating tumor cells and exosomes in a single blood sample. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the microfluidic device provided by the present invention.

[0045] Figure 2 This is a top view of the microfluidic device provided by the present invention.

[0046] Figure 3 This is an exploded view of the microfluidic device provided by the present invention.

[0047] Figure 4 This is another exploded view of the microfluidic device provided by the present invention.

[0048] Figure 5This is a cross-sectional view of the enrichment seat and the limiting boss provided by the present invention.

[0049] Figure 6 This is a schematic diagram of the enrichment seat and limiting boss provided by the present invention.

[0050] Figure 7 This is a schematic diagram of the enrichment module.

[0051] Figure 8 It is a microfluidic chip mask.

[0052] Figure 9 It is a microfluidic test bench.

[0053] Figure 10 It is a collaborative Dean flow inertial focusing sorting outlet trajectory diagram.

[0054] Figure 11 This is a schematic diagram of a three-dimensional model of exosome enrichment using a pit-assisted thermal field.

[0055] Figure 12 The concentration distribution diagrams are shown for (a) cylindrical pit chambers, (b) elliptical arc-shaped pit chambers, and (c) chambers without pits when equilibrium is reached.

[0056] Figure 13 This is a schematic diagram showing the maximum enrichment concentration multiple of particles in different chambers within 60 minutes.

[0057] Figure 14 This is a schematic diagram showing the convection velocity, thermophoretic velocity, and the combined velocity of the two at the center of the (a) pit and the edge of (b) in different chambers.

[0058] Figure 15 This is a schematic diagram of the deterministic lateral displacement principle.

[0059] Figure 16 It is a cloud map of electric field intensity distribution.

[0060] Figure 17 This is a schematic diagram showing the motion of two types of particles in DLD arrays with and without electric fields.

[0061] Figure 18 This is a particle trajectory diagram of a 6μm particle under different conditions.

[0062] In the diagram, 1 is a fixing plate; 11 is the first sorting module; 111 is a curved microchannel; 112 is a rectangular cavity; 12 is an enrichment module; 121 is the first channel; 122 is the enrichment cavity; 123 is a laser; 124 is a top cone; 125 is a circular hole; 126 is an enrichment seat; 127 is a limiting cavity; 128 is an annular washer; 13 is the second sorting module; 131 is a sorting cavity; 132 is the second channel; 133 is the normal blood cell outlet; 134 is the circulating tumor cell outlet; 135 is a microcolumn; 136 is an electrophoresis plate; 137 is a flow channel; 138 is the second normal blood cell outlet; 139 is an AC power supply; 14 is a sheath fluid input module; 141 is a sheath fluid channel; 2 is a substrate; and 21 is a limiting boss. Detailed Implementation

[0063] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0064] like Figure 1 , Figure 3 and Figure 4 The microfluidic device shown can sort circulating tumor cells and enriched exosomes, including a fixing plate 1 and a base plate 2 located below the fixing plate 1. A limiting boss 21 is provided on the base plate 2, and a limiting cavity 127 is provided on the lower side of the fixing plate 1 to cooperate with the limiting boss 21. When the fixing plate 1 is attached to the base plate 2, the limiting boss 21 extends into the limiting cavity 127 to limit the relative position of the fixing plate 1 and the base plate 2.

[0065] like Figure 2 As shown, the fixed plate 1 is provided with a first sorting module 11 for separating cells and exosomes, an enrichment module 12 for enriching exosomes, and a second sorting module 13 for sorting normal blood cells and circulating tumor cells. The inlet of the enrichment module 12 is connected to the exosome outlet of the first sorting module 11, and the inlet of the second sorting module 13 is connected to the cell outlet of the first sorting module 11. The inlet of the sorting module is also provided with a sheath fluid input module 14 for inputting sheath fluid into the second sorting module 13.

[0066] like Figure 2 As shown, the enrichment module 12 includes a first channel 121 horizontally disposed within the fixed plate 1, an enrichment cavity 122 disposed at the bottom of the first channel 121 with a circular cross-section, and a laser 123 located directly above the enrichment cavity 122 for heating cell exosomes. The heating area of ​​the laser 123 is cylindrical and centered with the enrichment cavity 122. The outer diameter of the heating area is smaller than the outer diameter of the enrichment cavity 122. The bottom of the enrichment cavity 122 is provided with a structure to enhance the synergistic effect of thermophoresis and thermal convection.

[0067] The outer diameter of the enrichment cavity 122 is greater than or equal to the width of the first channel 121. The fixing plate 1 is made of a transparent material, such as biocompatible silicone PDMS or plexiglass. The laser emitted by the laser 123 irradiates the heating area from top to bottom, rapidly and precisely heating the suspension flowing into the enrichment cavity 122. This better utilizes the synergistic effect of thermophoresis and thermal convection, increasing the temperature gradient of the enrichment cavity 122, enhancing the thermophoretic effect, and enriching cell exosomes in the enrichment cavity 122 more quickly and efficiently.

[0068] like Figure 5-7 As shown, the structure used to enhance the synergistic effect of thermophoresis and thermal convection is a top cone 124 located at the bottom of the enrichment cavity 122. The top cone 124 is centered on the enrichment cavity 122, and the generatrix of the top cone 124 is an arc. The outer diameter of the top cone 124 gradually decreases from bottom to top. The intersection of the longitudinal section passing through the centerline of the top cone 124 and the outer surface of the top cone 124 is the generatrix of the top cone 124. That is, the outer surface of the top cone 124 is an arc surface, which can enhance the synergistic effect of thermophoresis and thermal convection, thereby increasing the enrichment efficiency and enrichment factor.

[0069] In this embodiment, the outer surface of the top cone 124 is an elliptical arc surface with the concave side facing upwards.

[0070] like Figure 7 As shown, the bottom of the first channel 121 has a circular hole 125 that extends downward through the fixing plate 1. An enrichment seat 126 is sealed and fitted inside the circular hole 125. An enrichment cavity 122 is centered on the enrichment seat 126, and the enrichment seat 126 is mounted on the limiting boss 21. The enrichment seat 126 and the circular through hole are detachably and sealed, facilitating the removal of enriched cell exosomes for subsequent detection. The substrate 2 is attached to the lower side of the fixing plate 1, and the limiting boss 21 and the limiting cavity 127 cooperate to position the enrichment seat 126. When the limiting boss 21 is engaged in the limiting cavity 127, the enrichment seat 126 is engaged within the circular hole 125.

[0071] To improve sealing, an annular washer 128 is provided between the enrichment seat 126 and the fixing plate 1.

[0072] To achieve complete separation of blood cells, a curved microchannel 111 with a rectangular expansion and contraction array arranged on the inner side of the channel is proposed, such as... Figure 2 As shown, the first sorting module 11 includes a curved microchannel 111 disposed in the fixed plate 1 and a rectangular cavity 112 disposed on the curved inner side of the curved microchannel 111. There are several rectangular cavities 112 and they are evenly arranged along the length direction of the curved microchannel 111. The several rectangular cavities 112 are connected to the curved microchannel 111. The cell outlet of the first sorting module 11 is connected to the curved inner side of the curved microchannel 111, and the exosome outlet of the first sorting module 11 is connected to the curved outer side of the curved microchannel 111.

[0073] like Figure 2 As shown, the second sorting module 13 includes a sorting cavity 131 disposed in the fixed plate 1, a second channel 132 communicating with the sorting cavity 131, a normal blood cell outlet 133 and a circulating tumor cell outlet 134. The inlet of the second channel 132 is connected to the cell outlet of the first sorting module 11. A plurality of arrayed micropillars 135 are erected in the sorting cavity 131. The plurality of micropillars 135 are arranged obliquely along the flow direction of the sheath fluid in the second channel 132. The normal blood cell outlet 133 is located at the end of the normal blood cell movement direction, and the circulating tumor cell outlet 134 is located at the end of the circulating tumor cell movement direction.

[0074] The second sorting module 13 utilizes deterministic lateral displacement technology to separate normal blood cells from circulating tumor cells. This technology sorts cells based on particle size and shape. Its principle involves arranging micropillars 135 at a certain angle along the flow direction. Different critical diameters are generated depending on parameters such as the shape and spacing of the micropillars 135. This causes circulating tumor cells with a diameter larger than the critical diameter and a small number of normal blood cells to move towards the circulating tumor cell outlet 134, while normal blood cells with a diameter smaller than the critical diameter move to the normal blood cell outlet 133 and are discharged from there.

[0075] Among them, the micropillars are insulating micropillars.

[0076] like Figure 2 As shown, the sorting chamber 131 contains two opposing electrophoresis plates 136, with a flow channel 137 formed between the two electrophoresis plates 136. There are two circulating tumor cell outlets 134, each located close to the inner side of a different electrophoresis plate 136. A second outlet 138 for normal blood cells is located between the two circulating tumor cell outlets 134. Figure 1 As shown, the microfluidic device also includes an AC power supply 139, the negative terminal of which is connected to the second outlet 138 of normal blood cells, and the positive terminal of which is connected to the second channel 132.

[0077] The second sorting module 13 uses deterministic lateral displacement (DLD) technology in conjunction with insulator-induced dielectrophoresis (iDEP) to separate normal blood cells from circulating tumor cells flowing toward the circulating tumor cell outlet 134.

[0078] like Figure 2 As shown, the sheath fluid input module 14 includes four sheath fluid channels 141 that are respectively connected to the second channel 132, and the positive terminal of the AC power supply 139 is connected to one of the sheath fluid channels 141.

[0079] The working process of this microfluidic device is as follows:

[0080] A single whole blood sample to be tested is treated with red blood cell lysis buffer, centrifuged, and the supernatant is collected. The supernatant is pumped into the first sorting module 11 through the inlet of the first sorting module 11 using a pressure pump. The sample solution passes through a curved microchannel 111 with a rectangular expansion and contraction array. The Dean secondary flow induced by the curved microchannel 111 and the Dean flow caused by the array of several rectangular cavities 112 work together to enable micron-sized cells to be single-line focused on the inner side of the curve near the microchannel and sorted to the cell outlet. Nano-sized exosomes are not affected by the Dean flow and are uniformly dispersed in the channel and sorted to the exosome outlet.

[0081] The cell solution enters the second sorting module 13, where it forms a focused flow of three-dimensional monodisperse particles under the action of the sheath fluid. These particles sequentially enter the array region of microcolumns 135. In this region, smaller cells (normal blood cells) flow in a zigzag pattern and migrate laterally to the normal blood cell outlet 133. Larger blood cells and circulating tumor cells will undergo a collision flow under the action of deterministic lateral displacement, gradually migrating towards the circulating tumor cell outlet 134 along the direction of the array's staggered gradient.

[0082] During the migration of circulating tumor cells to the exit 134, the non-uniform electric field formed between the insulating micropillars 135 will cause the cells to be subjected to a dielectrophoretic force. This dielectrophoretic force will reduce the critical diameter for deterministic lateral displacement. Therefore, we can increase the gap between the micropillars 135 to prevent cells from clogging the micropillar 135 array.

[0083] Between the two electrophoresis plates 136, due to the difference in dielectric properties, normal blood cells are subjected to negative dielectric force, while circulating tumor cells are subjected to positive dielectric force, causing the circulating tumor cells to migrate to the circulating tumor cell outlets 134 on both sides and be collected.

[0084] After the exosomes enter the enrichment module 12, the cylindrical enrichment cavity 122 in the first channel 121 is gradually filled with the suspension as the exosome suspension is injected. As the suspension containing exosomes is continuously injected, the exosomes in the enrichment cavity 122 gradually migrate towards the pit at the bottom center of the cavity under the combined action of thermophoresis and thermal convection.

[0085] An air inlet is also provided at the entrance of the curved microchannel 111. The entrance of the curved microchannel 111 and the air inlet are opened selectively. After all the supernatant is injected, a certain amount of gas is injected through the air inlet at the same flow rate until the cell solution in the fixation plate 1 is completely discharged.

[0086] After enrichment, the enrichment seat 126 is separated from the through-well, and the enriched cell exosomes are collected from the enrichment cavity 122 for subsequent biomedical detection (PCR, immunochemical analysis, enzyme analysis, etc.).

[0087] To achieve optimal co-focusing separation, we used computer CFD simulation software to design and optimize our microchannel structure, enabling different types of blood cells to reach the same focusing position and achieve cell co-focusing, so as to completely separate the suspension containing blood cells. The superior performance of the proposed structure was verified through experiments.

[0088] The first sorting module 11 (microfluidic chip) was fabricated using soft photolithography: a 3-inch silicon wafer was selected as the mold substrate for the microfluidic chip. SU8 photoresist (GM1070 SU8 photoresist, Gresteltec Sarl, Switzerland) was spin-coated onto the smooth surface of the silicon wafer using a spin coater (SC100 type, BestTools, USA), and then heated on a heated plate (HS40 type, Terrey Pine Scientific, USA). After the above steps were completed, a film mask with a microchannel pattern (…) was… Figure 8 The photoresist is placed on a silicon wafer after pre-baking and then exposed using a photolithography machine (URE2000 / 35, Institute of Optics and Electronics, Chinese Academy of Sciences). After reheating the silicon wafer, the unexposed areas of photoresist are removed using a developer, resulting in a chip mold with a microfluidic structure. Finally, PDMS and its curing agent are mixed in a 10:1 ratio, poured into the silicon wafer mold, and baked to cure. Holes are then drilled and bonded onto the cured PDMS to ultimately fabricate a microfluidic chip.

[0089] To ensure stable suspension of the particles in the solution, glycerol and deionized water were prepared at a ratio of 2.2:7.8 to obtain a glycerol-water mixed solution. To prevent particle aggregation and adhesion to the walls, Tween 20 was added to the above mixed solution at 0.01% w / v. Polystyrene particles of 5, 10, and 15 μm (specification: 10 mL, 2.5 wt% polystyrene microsphere solution) were selected to simulate platelets, red blood cells, and white blood cells, respectively. The prepared solution was ultrasonically mixed for 10 min and then placed in a syringe, which was installed in the injection pump and connected to the chip channel inlet through a capillary tube. The chip was placed on a microscope platform, and a high-speed camera (Fastec, Germany) was mounted on the microscope (Chongqing Aote Optical Instrument Co., Ltd.) to photograph the high-speed movement of the particles on the chip channel.

[0090] To ensure the validity of the results, Figure 9Experiments were conducted on the microfluidic test bench shown. More than 2000 images were captured using a high-speed camera, and ImageJ software was used for trajectory overlay processing to obtain a superimposed streamline map of particle motion trajectories, ensuring sufficient particle distribution data for calculation. A detection line across the channel width was plotted in the images, and the grayscale distribution curve along this line was obtained using the Plot profile function to quantitatively describe and analyze the focusing width and focusing position of the particles. The closer the focusing position is to the sidewall, the smaller the focusing width, indicating better filtration performance of the channel for particles.

[0091] At the exit of the microchannel, we used a high-speed camera with a shutter speed of 5ms to continuously capture more than 2000 images. We then used ImageJ software to overlay the trajectories, resulting in a streamline diagram of the particle motion trajectory. Figure 10 As shown (micron-sized particles are all focused on the same focal center and are fully focused): at the exit of the curved microchannel 111, 5µm, 10µm, and 15µm particles are all focused into a straight line near the inner side of the microchannel, achieving full-peak focusing. This demonstrates the superior performance of our proposed synergistic Dean flow inertial sorting microchannel, which can be used to completely separate suspensions containing blood cells.

[0092] To achieve the synergistic effect of thermophoresis and convection in enriching exosomes, this patent proposes designing a pit structure at the bottom of the chamber to enhance the enrichment concentration and rate. The results of different pit structures are numerically simulated using computational fluid dynamics (CFD) software.

[0093] 1-1. Establishing a physical model:

[0094] Figure 11 This is a schematic diagram of a three-dimensional model of a pit-assisted thermal field microfluidic chip. The chip consists of an upper chip (fixed plate 1) and a lower chip (substrate 2). The upper chip is made of polydimethylsiloxane, and the lower part is assembled from the lower chip made of sapphire material. A laser beam passes through the upper PDMS chip and the cylindrical cavity sequentially from top to bottom along the central axis, with the laser focusing at the center of the pit at the bottom of the cavity's central axis. Considering the axisymmetry of the entire model, it can be simplified to a two-dimensional axisymmetric model.

[0095] The physical properties of the materials used in each part of the chip are shown in the table.

[0096] Table 1 Physical Properties

[0097]

[0098] 1-2. Governing equations and boundary conditions

[0099] Finite element method (FEM) simulation software was used to solve the governing equations for the continuous phase (base liquid) and the dispersed phase (particles) in the Euler coordinate system to simulate the solid-liquid two-phase flow of particles in a microscale chamber. The laser was a Gaussian beam that propagated downwards along the centerline to heat the liquid within the chamber. Laser parameters included a wavelength of 1480 nm, a beam radius of 20-300 μm, and a laser power of 10-1000 mW, which were obtained using the formula... The calculated Rayleigh distance is 1.32 mm, so it is assumed that the beam radius remains constant in the z-direction of laser propagation. However, in the r-direction, the laser power is coupled into the energy equation as a volumetric heat source through the Beer-Lambert model, causing a temperature gradient to form in the fluid in the vertical direction.

[0100] Laser power incident cross-section distribution:

[0101] Beer-Lambert Law:

[0102] I represents the light intensity, and k represents the absorption coefficient, with a value of 2649.0557.

[0103] Energy equation:

[0104] Where, ρ, C p Let k represent the fluid density, specific heat capacity, and thermal conductivity, respectively. T and u are the fluid temperature and velocity vectors, respectively. Q represents the volumetric heat source generated by the laser. Since the chip material under consideration has a low absorption rate for laser light, the absorption effect of the base fluid in the microcavity on laser energy is mainly considered.

[0105] The chip is surrounded by air, which is a boundary condition of kind II, with a room temperature of 25°C and a convective heat transfer coefficient of 2 W / m². -2 *K -1

[0106] Continuity equation:

[0107] Momentum equation:

[0108] In the above formula, P is the fluid pressure, μ is the fluid dynamic viscosity, and F... G This is the gravity of the fluid.

[0109] The walls of the microchamber are subject to no-slip boundary conditions. Within the cylindrical chamber, the number of particles in the suspension is very large. To ensure reasonable accuracy, the mass transfer equation is used to calculate the particle concentration distribution. Since the base liquid is affected by temperature gradients, resulting in natural convection, and the particles are affected by both temperature and concentration gradients, the governing equations include convection, thermophoresis, and concentration gradient terms. The corresponding governing equations are shown below:

[0110]

[0111] c represents the particle concentration, S T Let be the thermophoretic mobility coefficient. In this mass transfer equation, This refers to the speed of hot swimming. Let be the diffusion coefficient of the particles, and k be the Boltzmann constant.

[0112] 1-3 Numerical Simulation Results

[0113] We investigated the effect of different pit shapes on enrichment. In the numerical simulation results, we selected three representative pit structures (elliptical arc pit, cylindrical pit, and pitless chamber) for demonstration and analysis. The relevant simulation results are as follows:

[0114] Figure 12 This is a cloud map showing the concentration distribution within the chamber of a microfluidic chip with different pit structures. Figure 12 In (a) and (b), it can be seen that particles can be enriched in the pit at the bottom of the central axis of the chamber. Compared with the chamber without pits, the chamber with cylindrical pits reduces the maximum enrichment concentration factor of particles to 13200 times, which has a negative effect on the enrichment effect. However, in the chamber with elliptical arc-shaped pits, the maximum enrichment concentration factor is further increased to 18400 times.

[0115] from Figure 13 It can be seen that the maximum enrichment concentration factor increases fastest in the elliptical arc-shaped pit, while it increases slowest in the cylindrical pit. Taking the time to reach a maximum enrichment concentration factor of 2500 as the benchmark, the time required for the pitless chamber and the cylindrical pit chamber is 12 minutes; however, the elliptical arc-shaped pit chamber only requires 6 minutes. Therefore, among the three chamber types, the elliptical arc-shaped pit chamber has the fastest enrichment rate and the highest enrichment concentration, while the cylindrical pit chamber has the slowest enrichment rate and the lowest enrichment concentration.

[0116] exist Figure 14 In this study, the magnitudes of convection velocity, thermophoretic velocity, and combined velocity near the center and edges of the depression were analyzed. Figure 14As can be seen from the three velocities in the same chamber in (b), the convection velocity is relatively large, resulting in a large overall velocity. Therefore, convection plays a dominant role at the edge of the pit, and particles migrate mainly towards the center of the bottom surface of the chamber through convection in this region. In addition, the velocities of the elliptical arc-shaped pit chamber and the cylindrical pit chamber are greater than those of the chamber without pits, which proves that the addition of pits can increase the convection velocity. Among the three chambers, the thermophoretic velocities at the center of the pits in the elliptical arc-shaped pit chamber, the cylindrical pit chamber, and the chamber without pits are 10.21 μm / s, 8.34 μm / s, and 9.76 μm / s, respectively. The thermophoretic velocity of the cylindrical pit chamber is the largest. Therefore, the elliptical arc-shaped pit chamber has the largest enrichment factor for particles and the shortest enrichment time. This elliptical arc-shaped pit result is the optimal structure.

[0117] 2-1. Deterministic Lateral Displacement Theory

[0118] Deterministic Lateral Displacement (DLD) technology is a sorting technique based on particle size and shape. Its principle involves arranging micropillars 135 at a certain angle along the flow direction. Different critical diameters are generated depending on parameters such as the column shape and spacing of the micropillars 135. The definition of the critical diameter is as follows: Figure 15 As shown, G is the gap between micropillars 135, D is the cross-sectional diameter of micropillars 135, λ is the spacing between micropillars 135, Δλ is the displacement, the displacement fraction ε=Δλ / λ, the tilt angle between the micropillar 135 array and the mainstream direction is α, and ε=tanα. The left figure shows that the period N=1 / ε=4, and the width of each first channel through a single gap is β, which is half of the critical diameter of the channel. When the particle size is larger than the critical diameter, the particles will undergo a "bump" motion mode, and when the particle size is smaller than the critical diameter, the particles will undergo a "zigzag" flow. (Reference: Zeming KK, Ranjan S, Zhang, Y. Rotational separation of non-spherical bioparticles using I-shaped pillar arrays in a microfluidic device[J].NatureCommunications,2013,4(1):1625.)

[0119] 2-2. The Theory of Insulating Wall Induced Dielectrophoresis (iDEP)

[0120] iDEP, also known as electrodeless DEP or insulating wall induced dielectrophoresis DEP, is based on the electroosmotic flow theory. A double electric layer is generated at the solid-liquid interface where the channel wall is located. Under the influence of an external electric field, ions in the fluid undergo directional movement, causing surrounding liquid molecules to move along with them. Traditional iDEP chips are not driven by an external pressure source (flow field) but only by electroosmosis. (Reference: Liang L, Qian S, Xuan X. Three-dimensional electrokinetic particle focusing in a rectangular microchannel[J]. Journal of Colloid and Interface Science, 2010, 350(1):377–379)

[0121] 2-3. Governing equations and boundary conditions

[0122] Particle trajectory tracking model:

[0123] In microfluidic chips, particles or cells are typically suspended in a buffer solution. During their movement, both the fluid and the external force field exert forces on the particles. The equation of motion for a single particle is expressed using the Lagrange method as follows:

[0124]

[0125] In the left-hand side of the above equation, x p The position vector of the particle, m p Let F represent the particle mass, and t represent relative time. On the right-hand side of the equation, F... f F represents the force exerted on a particle by a fluid. out This represents the force and electric field exerted on a particle by an external force field:

[0126] When an electric field is applied to a microchannel, its internal electric field distribution is determined by the Laplace equation, as follows:

[0127]

[0128] Where φ is the electric potential. The electric field strength is determined by the potential distribution.

[0129] In a non-uniform electric field, the different degrees of polarization inside the particle lead to uneven forces, thus subjecting it to the action of the electric field force.

[0130] When a particulate suspension is placed in a non-uniform electric field, the particles will undergo directional movement within the fluid due to the difference in polarization between the particles and the fluid. The degree of polarization is influenced by the conductivity and dielectric constant of the medium. In a non-uniform electric field, the expression for the dielectric force acting on suspended particles is:

[0131]

[0132] Where dp is the diameter of the particle. It is the equivalent dielectric constant of the medium. This represents the gradient of the squared electric field strength, K(ω) is the Clausius-Mossotti (CM) factor, and Re[K(ω)] is the real part of the CM factor. The direction of the dielectrophoretic force is determined by Re[K(ω)]. When Re[K(ω)] > 0, the dielectrophoretic force points towards the high field strength region; when Re[K(ω)] < 0, the dielectrophoretic force points towards the low field strength region. The expression for the CM factor is:

[0133]

[0134] in, Dielectric constants of the particle and the solution, respectively.

[0135] Flow field:

[0136] In microfluidic chips, suspended particles are typically moved by a fluid. Therefore, only the force generated by the fluid moving the particles needs to be considered. The forces to be considered are as follows:

[0137] When a particle moves in a viscous fluid, the velocity difference between the fluid and the particle causes a drag force that pulls the particle along with the fluid, known as the Stokes drag force, expressed as follows:

[0138] F D =6πμr p (U f -U p )

[0139] Where μ is the hydrodynamic viscosity, U f and U p These represent the velocities of the fluid and the particles, respectively.

[0140] Fluids in microchannels typically flow in a Poisson's flow pattern. Particles moving within this flow experience a force due to the velocity gradient, known as shear lift, expressed as:

[0141]

[0142] f LS This is a dimensionless lift coefficient. Besides shear gradient lift, particles are also affected by the channel walls. Lift caused by the walls generally includes two types. One is Saffman lift; the presence of the wall creates a velocity gradient, and when the particle is close to the wall, the wall provides additional drag. The lift generated by the shear-slip interaction between the particle and the wall is called Saffman lift, expressed as:

[0143]

[0144] Where K = 81.2, and γ is the fluid velocity gradient shear rate. Another type is wall lift. For particles farther from the wall, the shear gradient lift will cause them to move towards the wall. The wall will generate a wall lift force that inhibits the particles from moving towards it, expressed as:

[0145]

[0146] Where fLW is the lift coefficient. In DLD chips, the interaction between particles and the wall is mainly shear-slip, so Saffman lift is used to characterize the interaction between particles and the wall.

[0147] We established a particle trajectory tracking model using finite element method software to verify the particle motion behavior under the combined influence of fluid flow and electric field.

[0148] 2-4. Results and Discussion

[0149] When a voltage is applied to the inlet and outlet of a traditional micro-pillar 135 array microchannel (DLD), a non-uniform electric field is generated between the insulating micro-pillars 135 perpendicular to the mainstream direction. The electric field intensity distribution is as follows: Figure 16 As shown, the electric field intensity between the micropillars 135 perpendicular to the mainstream direction is symmetrically distributed. The electric field intensity between the micropillars 135 is larger closer to the wall of the micropillar 135 and smaller closer to the center of the channel. The electric field intensity distribution upstream and downstream of the micropillars 135 is exactly the opposite, with the electric field intensity being smaller closer to the wall of the micropillar 135 and larger further away from the wall of the micropillar 135.

[0150] Subsequently, in the aforementioned electric and flow fields, we released particles with sizes of 5 μm and 8.6 μm, respectively, along the focused centerline. Based on the empirical formula for critical diameter derived by Inglis et al., we calculated the critical diameter in the absence of an electric field to be 10 μm (Reference: Inglis DW, Davis JA, Austin RH, et al. Critical particlesize for fractionation by deterministic lateral displacement[J]. Lab on aChip,2006,6(5):655).

[0151] The above situation was numerically simulated using the validated simulation model, and the simulation results are as follows: Figure 17As shown, in the absence of an electric field, the red streamlines represent the trajectory of 8.6 μm particles, and the blue streamlines represent the trajectory of 5 μm particles. Both particle sizes are smaller than the critical diameter of 10 μm, so when the microchannel is not energized, they exhibit zigzag flow. When an electric field is applied to the microchannel, the 8.6 μm particles begin to collide, while the 5 μm particles exhibit zigzag flow. This phenomenon indicates that applying an electric field to the micropillar 135 array can reduce the critical diameter through electrophoretic force. This phenomenon can be used to optimize the structure of the micropillar 135 array and / or its synergistic effect in sorting particles.

[0152] Furthermore, we observed the trajectory behavior of 6 μm particles in microchannels with different micropillar structures to cross-validate the accuracy of our above conclusions. The numerical simulation results are as follows: Figure 18 As shown, it can be observed that the critical diameter decreases with increasing voltage. When the voltage is 5V, the critical diameter is smaller and closer to that in the case of no electric field. When the voltage is 10V, the critical diameter decreases significantly, indicating that dielectric force can effectively reduce the critical diameter of the DLD device.

[0153] By applying voltages at the inlet and outlet of the DLD array chip, a non-uniform electric field is generated through insulating micropillars 135, causing the particles to be subjected to dielectric force within the DLD chip. Our research shows that the diameter of the DLD chip integrated with iDEP can be reduced, and the critical diameter of the iDEP-DLD decreases with increasing voltage and increases with increasing flow rate.

[0154] This phenomenon can be used to optimize the structure of traditional DLD chips. A DLD chip design integrating iDEP is proposed, bringing two beneficial effects:

[0155] 1. In DLD chip design, we can take advantage of the characteristic that the critical diameter of the DLD structure decreases after an electric field is applied. Combined with the influence of the micropillar 135 gap of the DLD chip itself on the critical diameter, we can increase the micropillar 135 gap of the DLD chip without changing the original critical diameter, thereby improving or even solving the shortcoming of traditional DLD chips being prone to clogging.

[0156] 2. In the actual process of sorting cells or particles, there is a definite critical diameter after the micro-pillar 135 structure microchannel has been processed. Therefore, traditional DLD chips cannot flexibly adjust the critical diameter of the micro-pillar 135 array according to the actual sorting conditions. However, the DLD chip we proposed that integrates iDEP can change the critical diameter of the micro-pillar 135 array by adjusting the applied voltage and flow rate according to the actual sorting conditions, so as to achieve multiple uses with one chip.

[0157] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A microfluidic device capable of sorting circulating tumor cells and enriched exosomes, characterized in that, The system includes a fixing plate (1), which contains a first sorting module (11) for separating cells and exosomes, an enrichment module (12) for enriching exosomes, and a second sorting module (13) for sorting normal blood cells and circulating tumor cells. The inlet of the enrichment module (12) is connected to the exosome outlet of the first sorting module (11), and the inlet of the second sorting module (13) is connected to the cell outlet of the first sorting module (11). 3) The inlet is also provided with a sheath fluid input module (14) for inputting sheath fluid into the second sorting module (13); the enrichment module (12) includes a first channel (121) horizontally disposed in the fixed plate (1), an enrichment cavity (122) disposed at the bottom of the first channel (121) and having a circular cross-section, and a laser (123) located directly above the enrichment cavity (122) for heating the suspension in the enrichment cavity (122). The heating area of ​​the laser (123) is cylindrical and is located above the enrichment cavity (122). 22) The heating area is centered and its outer diameter is smaller than that of the enrichment cavity (122). The bottom of the enrichment cavity (122) is provided with a structure to enhance the synergistic effect of thermophoresis and thermal convection. The structure to enhance the synergistic effect of thermophoresis and thermal convection is a top cone (124) at the bottom of the enrichment cavity (122). The top cone (124) is centered and its generatrix is ​​an arc. The bottom of the first channel (121) has a circular shape that extends downward through the fixing plate (1). The hole (125) is sealed with an enrichment seat (126), and the enrichment cavity (122) is centered on the enrichment seat (126); a base plate (2) is provided below the fixing plate (1), and a limiting boss (21) is provided on the base plate (2). The enrichment seat (126) is provided on the limiting boss (21). A limiting cavity (127) that cooperates with the limiting boss (21) is provided on the lower side of the fixing plate (1), and the hole (125) is provided in the limiting cavity (127).

2. The microfluidic device for sorting circulating tumor cells and enriching exosomes according to claim 1, characterized in that, The first sorting module (11) includes a curved microchannel (111) disposed in the fixed plate (1) and a rectangular cavity (112) disposed on the curved inner side of the curved microchannel (111). The rectangular cavity (112) is a plurality of such cavities and is uniformly arranged along the length direction of the curved microchannel (111). The plurality of rectangular cavities (112) are connected to the curved microchannel (111). The cell outlet of the first sorting module (11) is connected to the curved inner side of the curved microchannel (111), and the exosome outlet of the first sorting module (11) is connected to the curved outer side of the curved microchannel (111).

3. The microfluidic device for sorting circulating tumor cells and enriching exosomes according to claim 1, characterized in that, The second sorting module (13) includes a sorting cavity (131) disposed in the fixed plate (1), a second channel (132) communicating with the sorting cavity (131), a normal blood cell outlet (133) and a circulating tumor cell outlet (134). The inlet of the second channel (132) is connected to the cell outlet of the first sorting module (11). A plurality of arrayed micropillars (135) are erected in the sorting cavity (131). The plurality of micropillars (135) are arranged obliquely along the flow direction of the sheath fluid in the second channel (132). The normal blood cell outlet (133) is located at the end of the normal blood cell movement direction, and the circulating tumor cell outlet (134) is located at the end of the circulating tumor cell movement direction.

4. The microfluidic device for sorting circulating tumor cells and enriching exosomes according to claim 3, characterized in that, The sorting chamber (131) is provided with two opposing electrophoresis plates (136), and a flow channel (137) is formed between the two electrophoresis plates (136). There are two circulating tumor cell outlets (134), which are respectively located close to the inner side of different electrophoresis plates (136). A second normal blood cell outlet (138) is provided between the two circulating tumor cell outlets (134). The microfluidic device also includes an AC power supply (139). The negative terminal of the AC power supply (139) is connected to the second normal blood cell outlet (138), and the positive terminal of the AC power supply (139) is connected to the second channel (132).

5. The microfluidic device for sorting circulating tumor cells and enriching exosomes according to claim 4, characterized in that, The sheath fluid input module (14) includes several sheath fluid channels (141) connected to the second channel (132), and the positive terminal of the AC power supply (139) is connected to one of the sheath fluid channels (141).

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