Microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes

Abstract

The invention provides a microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes. The device comprises a fixed plate, a first sorting module used for separating cells from cell exosomes, an enrichment module used for enriching the cell exosomes and a second sorting module used for sorting normal blood cells and circulating tumor cells are arranged in the fixed plate, an inlet of the enrichment module is communicated with an exosome outlet of the first sorting module, and an outlet of the enrichment module is communicated with an exosome outlet of the second sorting module. The inlet of the second sorting module is communicated with the cell outlet of the first sorting module, and the inlet of the sorting module is also provided with a sheath fluid input module for inputting sheath fluid into the second sorting module. According to the microfluidic device capable of sorting the circulating tumor cells and enriching the exosomes in the cells, the separation of the cells and the exosomes is realized by utilizing cooperative Dean flow microfluidic inertial sorting, and then the efficient enrichment of the exosomes is realized by utilizing a pit-assisted thermal field; and meanwhile, a deterministic lateral displacement technology (DLD) is combined with an insulation dielectrophoresis technology (i DEP) to realize separation of tumor cells and normal cells.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics technology and relates to a microfluidics device capable of sorting circulating tumor cells and enriched cell exosomes from a single blood sample. Background Art

[0002] Circulating tumor cells (CTCs) refer to tumor cells that enter the human peripheral blood. Monitoring the type and quantity of CTCs is of great significance for the patient's prognosis, efficacy evaluation and individualized treatment. The traditional detection method is to use flow cytometry to sort and count cells in blood samples. This method requires expensive experimental instruments, complex experimental operations, and consumes a large amount of reagents. In addition, the detection is time-consuming and expensive.

[0003] Exosomes refer to small vesicles that contain complex RNA and proteins. Cancer markers (exosomes or exosomes) 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 in the early stages of cancer is extremely low, and there are micron-sized cells such as white blood cells and red blood cells, which results in low sensitivity and accuracy of the instrument's detection of cellular 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-nanoparticles using microfluidic chips, realizing many functions of traditional laboratories such as separation and detection. Microfluidic chips have the advantages of high integration, fast processing speed, low reagent consumption, and low cost, which have brought about tremendous changes in detection technology and made integrated rapid detection of detection instruments possible.

[0005] In microfluidics, particle manipulation methods are divided into two categories: passive and active methods, depending on whether there is energy input. The passive method mainly relies on differences in particle size, shape, density, and other characteristics, as well as the inertial force and viscosity of the fluid, to achieve particle manipulation under a specific microchannel structure. The active method uses external fields (physical fields such as magnetic fields, acoustic fields, electric fields, and thermal fields) to manipulate particles in a solution. The magnetic field manipulation method requires modification of magnetic beads with specific antibodies to effectively improve the accuracy of manipulation. The preparation process of the particles is relatively complicated, and after the manipulation is completed, it is difficult to clean up the magnetic material, which affects the performance of the sample; the acoustic field manipulation method uses ultrasonic fields to induce acoustic flow and apply acoustic radiation force to the particles to migrate them to a specific position. This method is simple to process on a chip, but it is large in size and difficult to miniaturize; the light field manipulation method: using focused lasers to apply scattering force and gradient force to the particles for manipulation, which requires complex and costly platform equipment.

[0006] Thermophoresis manipulation of particles can be divided into static and flowing types. Traditional thermophoresis manipulation is generally static and has generally low efficiency, while flowing thermophoresis can be combined with the thermal convection effect to make thermal field manipulation of particles more efficient. However, general flowing thermophoresis enrichment often has unreasonable structural design, cannot make good use of the synergistic effect of thermophoresis and thermal convection, and has disadvantages such as difficulty in collecting enriched particles.

[0007] Dielectrophoresis manipulation technology can perform continuous and precise manipulation of unlabeled cells during flow, and can be divided into ordinary dielectrophoresis technology, insulator-induced dielectrophoresis (iDEP) technology, and non-contact dielectrophoresis (cDEP). Ordinary dielectrophoresis and non-contact dielectrophoresis require the production of 3D electrodes, making the chip structure complex. In addition, traditional insulator-induced dielectrophoresis technology generally only uses electroosmotic flow as the driving force, and the flux is low.

[0008] In addition, both circulating tumor cells and exosomes are important markers for early diagnosis of cancer. Traditional microfluidic processing methods often focus on enriching one of them, while treating the other cell marker as waste, which greatly reduces the accuracy of early cancer diagnosis. Therefore, a microfluidic solution that can sort circulating tumor cells and enrich exosomes from a single blood sample is urgently needed. Summary of the invention

[0009] The purpose of the present invention is to address the above-mentioned problems in the existing technology and to propose a low-cost, high-efficiency microfluidic device that can sort circulating tumor cells and enrich cell exosomes.

[0010] The purpose of the present invention can be achieved by the following technical solutions:

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

[0012] The single whole blood sample to be tested is treated with red blood cell lysis solution, and the supernatant is taken after centrifugation. The supernatant is pumped into the inlet of the first sorting module by a pressure pump, and the cells and cell exosomes are sorted to different outlets through the first sorting module. The cells enter the second sorting module, and normal blood cells and circulating tumor cells are sorted out under the action of the second sorting module; the cell exosomes enter and are enriched in the enrichment module.

[0013] In the above-mentioned microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes, the enrichment module comprises a first channel horizontally arranged in a fixed plate, an enrichment cavity with a circular cross-section arranged at the bottom of the first channel, and a laser located directly above the enrichment cavity for heating the suspension in the enrichment cavity. The heating area of ​​the laser is cylindrical and arranged in the center of the enrichment cavity. 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 increasing 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 silica gel or organic glass, to facilitate transmission of the laser into the enrichment cavity.

[0015] The laser emitted by the laser passes through the transparent fixed plate from top to bottom, and then irradiates the heating area to quickly and accurately heat the suspension, forming a temperature gradient field, causing the cellular exosomes in the suspension to migrate to the center of the enrichment cavity.

[0016] In the above-mentioned microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes, the structure for increasing the synergistic effect of thermophoresis and thermal convection is a top cone arranged at the bottom of the enrichment chamber, the top cone is arranged in the center of the enrichment chamber, and the generatrix of the top cone is an arc. The outer diameter of the top cone gradually decreases from bottom to top, and the intersection line of the longitudinal section through the center line of the top cone and the outer surface of the top cone is the generatrix of the top cone, that is, the outer surface of the top cone is an arc surface, which can increase the synergistic effect of thermophoresis and thermal convection, and increase the enrichment efficiency and enrichment multiple.

[0017] The top cone can increase the temperature gradient in the enrichment chamber, enhance the thermophoresis effect, better utilize the synergistic effect of thermophoresis and thermal convection, and enrich the cell exosomes in the enrichment chamber faster and more efficiently.

[0018] Furthermore, the outer surface of the top cone is an elliptical arc surface, and the concave surface faces upward.

[0019] In the above-mentioned microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes, the bottom of the first channel has a circular hole penetrating downwardly through the fixed plate, an enrichment seat is sealed in the circular hole, and the enrichment chamber is centrally arranged on the enrichment seat.

[0020] A circular hole is set through the bottom of the first channel, and the enrichment seat can be detachably set in the circular hole, so that the enrichment seat can be easily taken out, thereby facilitating the collection of cell exosomes.

[0021] In the above-mentioned microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes, a base plate is provided below the fixed plate, a limiting boss is provided on the base plate, the enrichment seat is provided on the limiting boss, a limiting cavity cooperating with the limiting boss is provided on the lower side of the fixed plate, and the circular hole is provided in the limiting cavity.

[0022] The enrichment seat and the circular through hole are detachably sealed to facilitate the removal of enriched exosomes for subsequent testing. The base plate is attached to the lower side of the fixed plate, and the limiting boss cooperates with the limiting cavity to realize the positioning of the enrichment seat. When the limiting boss is engaged in the limiting cavity, the enrichment seat is engaged in the circular hole.

[0023] In order to improve the sealing performance, an annular gasket is arranged between the enrichment seat and the fixing plate.

[0024] In the above-mentioned microfluidic device capable of sorting circulating tumor cells and enriched cell exosomes, the first sorting module comprises a curved microchannel arranged in a fixed plate and a rectangular cavity arranged on the inner side of the curve of the curved microchannel, the rectangular cavities are in a plurality and are evenly 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 curve of the curved microchannel, and the exosome outlet of the first sorting module is connected to the outer side of the curve of the curved microchannel.

[0025] The curved microchannel extends in a curved horizontal direction. Under the synergistic effect of the Dean secondary flow induced by the curved microchannel and the Dean flow caused by several rectangular cavity arrays, micron-sized cells (blood cells and circulating tumor cells) are focused in a single line on the inner side of the curve close to the microchannel at almost the same focusing center. Nano-sized cell exosomes are evenly dispersed in the channel due to their small particle size and are almost unaffected by the Dean flow, thereby sorting cells and cell exosomes to different exits.

[0026] In the above-mentioned microfluidic device capable of sorting circulating tumor cells and enriched cell exosomes, the second sorting module comprises a sorting chamber arranged in a fixed plate, a second channel connected to the sorting chamber, a normal blood cell outlet and a circulating tumor cell outlet, the inlet end of the second channel is connected to the cell outlet of the first sorting module, a plurality of array-distributed microcolumns are vertically arranged in the sorting chamber, and the plurality of microcolumns are obliquely arranged 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 uses deterministic lateral displacement technology to separate normal blood cells from circulating tumor cells. Deterministic lateral displacement technology (DLD) is a technology for sorting based on particle size and shape. Its principle is to arrange microcolumns at a certain inclination along the flow direction. Different critical diameters will be generated according to different parameters such as the column shape and microcolumn gap of the microcolumns. Circulating tumor cells with a particle size larger than the critical diameter and a small amount of normal blood cells are moved toward the outlet of circulating tumor cells, and normal blood cells with a particle size smaller than the critical diameter are moved to the outlet of normal blood cells and discharged from the outlet of normal blood cells. Among them, the microcolumns are insulating microcolumns.

[0028] In the above-mentioned microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes, two electrophoresis plates arranged opposite to each other are provided in the sorting chamber, a flow channel is formed between the two electrophoresis plates, there are two circulating tumor cell outlets and they are respectively arranged close to the inner sides of different electrophoresis plates, and a second normal blood cell outlet is provided between the two circulating tumor cell outlets; the microfluidic device also includes an AC power supply, the negative pole of the AC power supply is connected to the second normal blood cell outlet, and the positive pole of the AC power supply is connected to the second channel.

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

[0030] In the above-mentioned microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes, the sheath fluid input module comprises a plurality of sheath fluid channels connected to the second channel, and the positive pole 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 solution, and the supernatant is taken after centrifugation. The supernatant is pumped into the first sorting module through the inlet of the first sorting module using a pressure pump, so that the sample solution passes through a curved microchannel with a rectangular contraction and expansion array. The Dean secondary flow induced by the curved microchannel and the Dean flow caused by several rectangular cavity arrays work synergistically to achieve single-line focusing of micron-sized cells at almost the same focusing center, and the cells are sorted to the cell outlet. Nano-sized cell exosomes are evenly dispersed in the channel due to their small particle size and are almost unaffected by the Dean flow, and the cell exosomes are sorted to the exosome outlet.

[0033] The cell solution enters the second sorting module, and under the action of the sheath fluid, it forms a focused flow of three-dimensional monodisperse particles, and enters the microcolumn array area in turn. In this area, cells with smaller particle sizes (normal blood cells) flow in a zigzag shape and migrate laterally to the normal blood cell outlet; blood cells with larger particle sizes and circulating tumor cells will flow in a "collision" shape under the action of deterministic lateral displacement, and gradually migrate along the direction of the array's staggered gradient toward the direction close to the circulating tumor cell outlet.

[0034] During the migration process toward the exit of circulating tumor cells, the cells are subjected to the dielectrophoretic force due to the inhomogeneous electric field formed between the insulating micropillars. This dielectrophoretic force reduces the critical diameter for deterministic lateral displacement. Therefore, we can increase the gap between the micropillars to prevent the cells from blocking the micropillar array.

[0035] Between the two electrophoresis plates, due to the difference in dielectric properties, normal blood cells will be affected by negative dielectrophoretic force, while circulating tumor cells will be affected by positive dielectrophoretic force, which will cause the circulating tumor cells to migrate to the circulating tumor cell outlets on both sides and be collected.

[0036] After the cellular exosomes enter the enrichment module, with the injection of the exosome suspension, the cylindrical enrichment cavity in the first channel is gradually filled with the suspension. As the suspension containing exosomes continues to be injected, the cellular exosomes in the enrichment cavity gradually migrate to the pit at the bottom of the 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 curved microchannel inlet 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 fixed plate is completely discharged.

[0038] After the enrichment is completed, the enrichment seat is separated from the through hole, 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 inner side of the bend of the microchannel, so that the Dean secondary flow induced by the curved microchannel and the rectangular cavity structures are in the same direction, synergistically strengthening the focusing of the particles, so that micron-level cells can achieve single-line focusing, and the focusing center position of micron-level cells of different particle sizes is the same, so as to achieve the purpose of completely separating micron-level cells, avoid the mutual influence of micron-level cells and nano-level cell exosomes, reduce the required focusing channel length, speed up the solution processing, and improve the detection rate of circulating tumor cells.

[0041] 2. The pit-assisted thermal field manipulation method is adopted. Compared with other active manipulation methods, it does not need to mark the particles, requires less energy, and does not harm the biological particles. Compared with the traditional method of using mechanical heating to form a thermal field, which causes problems such as complex chip structure and difficult processing, the present invention uses laser heating technology to quickly and accurately heat the enrichment cavity to form a constant temperature gradient field, thereby effectively manipulating cell derivatives; using a special elliptical top cone structure, the synergistic effect of thermophoresis and thermal convection is better utilized, the temperature gradient in the center of the enrichment cavity is increased, the thermophoresis effect is enhanced, and the cell exosomes are enriched faster and more efficiently; using a detachable design, the problem of difficulty in collecting enriched particles in traditional flow-type thermophoresis enrichment is solved.

[0042] 3. The second sorting area utilizes the synergistic effect of the microcolumn array and dielectric electrophoresis. In the microcolumn array, deterministic lateral displacement is used to sort cells of different diameters. In the migration process of deterministic lateral displacement, the non-uniform electric field formed between the microcolumn arrays can be used to increase the distance between the microcolumn arrays without changing the critical diameter, thereby solving the problem of easy clogging of the traditional microcolumn array. The difference in dielectric properties is used to migrate circulating tumor cells with a particle size not much different from that of normal blood cells toward the exit of circulating tumor cells, greatly improving the detection rate of circulating tumor cells. The electrodes are connected at the second channel to replace the traditional embedded three-dimensional electrodes. This sorting method has the advantages of high sorting efficiency, simple structure, and the sorted circulating tumor cells have intact biological activity.

[0043] 4. Compared with traditional methods, this device can simultaneously sort tumor cells and enrich cell exosomes, and separate the two in advance through microfluidics coordinated Dean flow inertial sorting, avoiding the cross-influence of the two on subsequent biomedical testing, greatly improving the early detection rate of cancer, and providing a simple, low-cost and accurate microfluidic manipulation device for detecting circulating tumor cells and cell exosomes in a single blood sample. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0046] Figure 3 Schematic diagram of an explosion of the microfluidic device provided by the present invention.

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

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

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

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

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

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

[0053] Fig.10 It is the exit trajectory diagram of the collaborative Dean flow inertial focusing sorting.

[0054] Fig.11 Schematic diagram of the three-dimensional model of pit-assisted thermal field enrichment of exosomes.

[0055] Fig.12 These are the concentration distribution diagrams of (a) cylindrical pit chamber, (b) elliptical pit chamber, and (c) no pit chamber when equilibrium is reached.

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

[0057] Fig.14 Schematic diagram of the convection velocity, thermophoresis velocity, and the combined velocity of the two at (a) the center and (b) the edge of the pit in different chambers.

[0058] Fig.15 It is the principle diagram of deterministic lateral displacement.

[0059] Fig.16 It is the electric field intensity distribution cloud diagram.

[0060] Fig.17 This is a schematic diagram of the movement of two types of particles in the DLD array with and without electric field.

[0061] Fig.18 It is the particle trajectory diagram of 6μm particles under different conditions.

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

[0063] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0064] like Figure 1 , Figure 3 and Figure 4 The microfluidic device for sorting circulating tumor cells and enriched cell exosomes shown includes a fixed plate 1 and a substrate 2 arranged below the fixed plate 1, a limiting boss 21 is provided on the substrate 2, and a limiting cavity 127 matched with the limiting boss 21 is provided on the lower side of the fixed plate 1. When the fixed plate 1 is attached to the substrate 2, the limiting boss 21 extends into the limiting cavity 127 to limit the relative position of the fixed plate 1 and the substrate 2.

[0065] like Figure 2 As shown, 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 are provided in the fixed plate 1. 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 arranged in the fixing plate 1, an enrichment chamber 122 with a circular cross-section arranged at the bottom of the first channel 121, and a laser 123 located directly above the enrichment chamber 122 for heating the cell exosomes. The heating area of ​​the laser 123 is cylindrical and is arranged in the center of the enrichment chamber 122. The outer diameter of the heating area is smaller than the outer diameter of the enrichment chamber 122. The bottom of the enrichment chamber 122 is provided with a structure for increasing the synergistic effect of thermophoresis and thermal convection.

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

[0068] like Figure 5-7 As shown, the structure for increasing the synergistic effect of thermophoresis and thermal convection is a top cone 124 disposed at the bottom of the enrichment chamber 122. The top cone 124 is arranged in the center of the enrichment chamber 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, and the intersection line of the longitudinal section through the center line 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 increase the synergistic effect of thermophoresis and thermal convection, thereby increasing the enrichment efficiency and the enrichment multiple.

[0069] In this embodiment, the outer surface of the top cone 124 is an elliptical arc surface, and the concave surface faces upward.

[0070] like Figure 7 As shown, the bottom of the first channel 121 has a circular hole 125 that passes through the fixed plate 1 downward, and an enrichment seat 126 is sealed in the circular hole 125. The enrichment cavity 122 is centrally arranged on the enrichment seat 126, and the enrichment seat 126 is arranged on the limiting boss 21. The enrichment seat 126 and the circular through hole are detachably sealed and connected, which is convenient for taking out the enriched extracellular vesicles for subsequent detection. The substrate 2 is attached to the lower side of the fixed plate 1, and the limiting boss 21 cooperates with the limiting cavity 127 to realize the positioning of the enrichment seat 126. When the limiting boss 21 is engaged in the limiting cavity 127, the enrichment seat 126 is engaged in the circular hole 125.

[0071] In order to improve the sealing performance, an annular gasket 128 is provided between the enrichment seat 126 and the fixing plate 1 .

[0072] In order to achieve complete separation of blood cells, a curved microchannel 111 is proposed with a rectangular expansion and contraction array arranged inside the channel, such as Figure 2 As shown, the first sorting module 11 includes a curved microchannel 111 arranged in the fixed plate 1 and a rectangular cavity 112 arranged on the inner side of the curve 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. Several rectangular cavities 112 are connected to the curved microchannel 111. The cell outlet of the first sorting module 11 is connected to the inner side of the curve of the curved microchannel 111, and the exosome outlet of the first sorting module 11 is connected to the outer side of the curve of the curved microchannel 111.

[0073] like Figure 2 As shown, the second sorting module 13 includes a sorting chamber 131 arranged in the fixed plate 1, a second channel 132 connected to the sorting chamber 131, a normal blood cell outlet 133 and a circulating tumor cell outlet 134, the inlet end of the second channel 132 is connected to the cell outlet of the first sorting module 11, and a plurality of array-distributed microcolumns 135 are vertically arranged in the sorting chamber 131, and the plurality of microcolumns 135 are obliquely arranged 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 uses deterministic lateral displacement technology to separate normal blood cells from circulating tumor cells. Deterministic lateral displacement technology is a technology for sorting according to particle size and shape. Its principle is to arrange microcolumns 135 at a certain inclination along the flow direction. Different critical diameters will be generated according to different parameters such as the column shape of the microcolumns 135 and the gap between the microcolumns 135. The circulating tumor cells with a particle size larger than the critical diameter and a small amount of normal blood cells are moved toward the circulating tumor cell outlet 134, and the normal blood cells with a particle size smaller than the critical diameter are moved to the normal blood cell outlet 133 and discharged from the normal blood cell outlet 133.

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

[0076] like Figure 2 As shown, two electrophoresis plates 136 are arranged opposite to each other in the sorting chamber 131, and a flow channel 137 is formed between the two electrophoresis plates 136. There are two circulating tumor cell outlets 134, which are arranged close to the inner sides of different electrophoresis plates 136, and a second normal blood cell outlet 138 is arranged between the two circulating tumor cell outlets 134. Figure 1 As shown, the microfluidic device further includes an AC power source 139 , the negative pole of the AC power source 139 is connected to the normal blood cell second outlet 138 , and the positive pole of the AC power source 139 is connected to the second channel 132 .

[0077] The second sorting module 13 uses deterministic lateral displacement technology (DLD) in conjunction with insulator-induced dielectrophoresis technology (iDEP) to separate normal blood cells and 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 respectively connected to the second channel 132 , and the positive pole of the AC power source 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 solution, and the supernatant is taken after centrifugation. The supernatant is pumped into the first sorting module 11 through the inlet of the first sorting module 11 by a pressure pump, so that the sample solution passes through the curved microchannel 111 with a rectangular contraction and expansion array. The Dean secondary flow induced by the curved microchannel 111 and the Dean flow caused by the array of several rectangular cavities 112 are synergistically used to achieve single-line focusing of micron-scale cells on the inner side of the curve close to the microchannel, and the cells are sorted to the cell outlet. Nanoscale cell exosomes are not affected by the Dean flow and are evenly dispersed in the channel, and the cell exosomes are sorted to the exosome outlet.

[0081] The cell solution enters the second sorting module 13, and forms a focused flow of three-dimensional monodisperse particles under the action of the sheath fluid, and enters the microcolumn 135 array area in turn. In this area, cells with smaller particle sizes (normal blood cells) flow in a zigzag shape and migrate laterally to the normal blood cell outlet 133; blood cells with larger particle sizes and circulating tumor cells will flow in a "collision" shape under the action of deterministic lateral displacement, and gradually migrate toward the circulating tumor cell outlet 134 along the direction of the array's staggered gradient.

[0082] During the migration process toward the circulating tumor cell exit 134, the cells are affected by the dielectrophoretic force due to the non-uniform electric field formed between the insulating micropillars 135. This dielectrophoretic force reduces the critical diameter of deterministic lateral displacement. Therefore, we can increase the gap between the micropillars 135 to prevent the cells from blocking the micropillar 135 array.

[0083] Between the two electrophoresis plates 136, due to the difference in dielectric properties, normal blood cells will be affected by negative dielectrophoretic force, while circulating tumor cells will be affected by positive dielectrophoretic 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, as the exosome suspension is injected, the cylindrical enrichment chamber 122 in the first channel 121 is gradually filled with the suspension. As the suspension containing exosomes is continuously injected, the exosomes in the enrichment chamber 122 gradually migrate to the pit at the bottom of the center of the chamber under the combined action of thermophoresis and thermal convection.

[0085] An air inlet is also provided at the entrance of the curved microchannel 111. Either the entrance of the curved microchannel 111 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 fixed plate 1 is completely discharged.

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

[0087] In order to achieve the optimal collaborative focusing separation effect, we use computer CFD simulation software to assist in the design and optimization of our microchannel structure, so that different types of blood cells can reach the same focusing position, realize cell confocusing, and completely separate the suspension containing blood cells. The superior performance of the proposed structure is verified through experiments.

[0088] The first sorting module 11 (microfluidic chip) was prepared by soft lithography: a 3-inch silicon wafer was selected as the mold substrate of the microfluidic chip, and SU8 photoresist (GM1070 SU8 photoresist, Gresteltec Sarl, Switzerland) was spin-coated on the smooth surface of the silicon wafer by a coating machine (SC100 coating machine, Best Tools, USA), and then placed on a heating plate (HS40 CNC heating plate, Terrey Pine Scientific, USA) for heating. After the above steps were completed, a film mask ( Figure 8 ) is placed on the pre-baked silicon wafer, and then exposed on a photolithography machine (URE2000 / 35 photolithography machine, Institute of Optical Technology, Chinese Academy of Sciences). After the silicon wafer is heated again, the photoresist of the unexposed part is removed by a developer to obtain a chip mold with a microfluidic structure. Finally, PDMS and its curing agent are mixed in a ratio of 10:1, and poured on the silicon wafer mold and baked and cured. The cured PDMS is further punched and bonded to finally make a microfluidic chip.

[0089] In order to make the particles stably suspended in the solution, glycerol and deionized water were prepared in a ratio of 2.2:7.8 to obtain a glycerol-water mixed solution. In order to prevent the particles from agglomerating and sticking to the wall, Tween 20 was added to the above mixed solution at 0.01% w / v. 5, 10, and 15 μm polystyrene particles (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 placed in a syringe after ultrasonic mixing for 10 minutes, and the syringe was installed in a syringe pump and connected to the chip channel inlet through a capillary. The chip was placed on a microscope platform, and a high-speed camera (Fastec, Germany) was installed on a microscope (Chongqing Aote Optical Instrument Co., Ltd.) to capture particles moving at high speed on the chip channel.

[0090] In order to ensure the validity of the results, Fig. 9The experiment was carried out on the microfluidic test bench shown in the figure. More than 2,000 images were taken with a high-speed camera, and the trajectory was superimposed using ImageJ software to obtain a streamline diagram of the particle motion trajectory to ensure that there was enough particle distribution data for calculation. In the above image, a detection line across the width of the flow channel was drawn, and the grayscale distribution curve on the line was obtained through the Plot profile function to quantitatively describe and analyze the focal width and focal position of the particles. The closer the focal position is to the side wall, the smaller the focal width is, indicating that the channel has a better filtering performance for particles.

[0091] At the exit of the microchannel, we used a high-speed camera with a shutter speed of 5ms to continuously take more than 2000 pictures, and used ImageJ software to superimpose the trajectories to obtain the particle motion trajectory superimposed streamline diagram as shown below: Fig.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, 5um, 10um, and 15um 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 the collaborative Dean flow inertial sorting microchannel we proposed, which can be used to completely separate the suspension containing blood cells.

[0092] In order to achieve the synergistic effect of thermophoresis and convection to enrich exosomes, this patent proposes to design a pit structure at the bottom of the chamber to increase the enrichment concentration and enrichment rate, and numerically simulate different pit results through computer computational fluid dynamics CFD software

[0093] 1-1. Establish physical model:

[0094] Fig.11 The figure is a schematic diagram of the three-dimensional model of the 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 combined with a lower chip made of sapphire. A laser beam passes through the upper PDMS chip and the cylindrical chamber from top to bottom along the central axis, and finally the focus of the laser is located at the center of the pit at the bottom of the central axis of the chamber. Considering that the entire model has axisymmetry, the model can be simplified into a two-dimensional axisymmetric model.

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

[0096] Table 1 Physical properties

[0097]

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

[0099] Finite element simulation software is used to solve the control equations of 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 is a Gaussian beam that propagates from top to bottom along the centerline to heat the liquid in the chamber. The laser parameters are a wavelength of 1480nm, a beam radius of 20-300um, and a laser power of 10-1000mW. The Rayleigh distance is calculated to be 1.32 mm, so it is assumed that the beam radius of the laser in the z direction remains unchanged. In the r direction, the laser power is coupled into the energy equation as a volume heat source through the Beer-Lambert model, so that the fluid produces a temperature gradient in the vertical direction.

[0100] Laser power incident cross-sectional distribution:

[0101] Beer-Lambert Law:

[0102] I is the light intensity and k is the absorption coefficient, which is 2649.0557.

[0103] Energy equation:

[0104] Among them, ρ, C p , k are the density, specific heat capacity and thermal conductivity of the fluid respectively. T and u are the temperature and velocity vector of the fluid respectively. Q represents the volume heat source generated by the laser. Since the absorption rate of the chip material under consideration to the laser is low, the absorption effect of the base liquid in the microchamber on the laser energy is mainly considered.

[0105] The chip is in contact with the air, which is the second type of boundary condition, where the room temperature is 25°C and the convection heat transfer coefficient is 2W*m -2 *K -1

[0106] Continuity equation:

[0107] Momentum equation:

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

[0109] The micro-chamber wall is a no-slip boundary condition. In the cylindrical chamber, the number of particles in the suspension is very large. To ensure rationality, the mass transfer equation is used to calculate the concentration distribution of the particles. Since the base liquid is affected by the temperature gradient, natural convection occurs, and the particles are affected by the temperature gradient and concentration gradient. Therefore, the control equation includes convection terms, thermophoresis terms, and concentration gradient terms. The corresponding control equation is as follows:

[0110]

[0111] c represents the concentration of particles, S T is the thermophoretic migration coefficient. In this mass transfer equation, is the thermophoresis velocity. is the diffusion coefficient of the particle, k is the Boltzmann constant

[0112] 1-3. Numerical simulation results

[0113] We studied the effects of different pit shapes on enrichment. In the numerical simulation results, we selected three representative pit structures (elliptical arc pits, cylindrical pits, and pit-free chambers) for display and analysis. The relevant simulation results are as follows:

[0114] Fig.12 This is a cloud diagram of the spatial concentration distribution in the chamber of a microfluidic chip with different pit structures. Fig.12 In (a) and (b), it can be seen that the particles can be enriched in the pit at the bottom of the chamber centerline. Compared with the chamber without pits, the chamber with cylindrical pits reduces the maximum enrichment concentration of particles to 13,200 times, which has a negative effect on the enrichment effect. In the chamber with elliptical arc pits, the maximum enrichment concentration is further increased to 18,400 times.

[0115] from Fig.13 It can be seen that the maximum enrichment concentration multiple of the elliptical arc pit increases the fastest, while the cylindrical pit increases the slowest. Taking the time for the maximum enrichment concentration multiple to reach 2500 times as the standard, the time required for the chamber without pits and the cylindrical pit chamber is 12 minutes, however, the elliptical arc pit chamber only takes 6 minutes. It can be seen that among the three chambers, the elliptical arc pit has the fastest enrichment speed and the highest enrichment concentration, while the cylindrical pit chamber has the slowest enrichment speed and the lowest enrichment concentration.

[0116] exist Fig.14 In the above figure, the convection velocity, thermophoresis velocity and comprehensive velocity near the center and edge of the pit are analyzed. Fig.14It can be seen from the three velocities of the same chamber in (b) that the convection velocity is larger, resulting in a larger integrated velocity. Therefore, convection plays a dominant role at the edge of the pit, and particles in this area migrate mainly to the center of the bottom surface of the chamber through convection. In addition, compared with the chamber without pits, the velocities of the elliptical arc pit chamber and the cylindrical pit chamber are larger, which proves that the setting of pits can increase the convection velocity. Among the three chambers, the thermophoresis velocities at the center of the pits of the elliptical arc pit chamber, the cylindrical pit chamber and the chamber without pits are 10.21μm / s, 8.34μm / s and 9.76μm / s, respectively. The thermophoresis velocity of the cylindrical pit chamber is the largest. Therefore, the elliptical arc pit chamber has the largest enrichment multiple for particles and the shortest enrichment time. This elliptical arc pit result is the optimal structure.

[0117] 2-1. Deterministic lateral displacement theory

[0118] Deterministic Lateral Displacement (DLD) technology is a technology for sorting particles based on their size and shape. The principle is to arrange micropillars 135 at a certain angle along the flow direction. Different critical diameters will be generated according to the different parameters such as the column shape and the gap between micropillars 135. The definition of critical diameter is as follows: Fig.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 micropillar 135 array is inclined at an angle α to the mainstream direction, and ε=tanα. The left figure shows that the period N=1 / ε=4, and the width of each first flow 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 have a "bump" motion mode, and when the particle size is smaller than the critical diameter, the particles will have 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]. Nature Communications, 2013, 4(1): 1625.)

[0119] 2-2. Insulating wall induced dielectrophoresis (iDEP) theory

[0120] iDEP is also called electrodeless DEP or insulating wall induced dielectrophoresis DEP. According to the electroosmotic flow theory, a double electric layer will be generated at the solid-liquid interface where the channel wall is located. Under the action of an external electric field, the ions in the fluid will move in a directional manner, driving the surrounding liquid molecules to move together. 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. Control equations and boundary conditions

[0122] Particle Trajectory Tracking Model:

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

[0124]

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

[0126] When an electric field is applied to a microchannel, its internal electric field distribution is determined by the Laplace equation, which is 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 particles lead to uneven forces, and thus they are affected by the electric field force.

[0130] When a particle suspension is in a non-uniform electric field, the particles will move in a directional manner in the fluid due to the different polarization degrees of the particles and the fluid, where the polarization degree is affected by the conductivity and dielectric constant of the medium. In a non-uniform electric field, the dielectrophoretic force on the suspended particles is expressed as:

[0131]

[0132] where dp is the diameter of the particle, is the equivalent dielectric constant of the medium, is 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 to the high field strength area, and when Re[K(ω)]<0, the dielectrophoretic force points to the low field strength area. The expression of the CM factor is:

[0133]

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

[0135] Flow field:

[0136] In microfluidic chips, suspended particles are usually driven by fluids, so only the forces generated by the fluid driving the particles need to be considered. The forces that need to be considered are as follows

[0137] When particles move in a viscous fluid, the velocity difference between the fluid and the particles causes the drag force on the particles to become the Stokes drag force, which is expressed as follows:

[0138] F D =6πμr p (U f -U p )

[0139] Where μ is the fluid dynamic viscosity, U f and U p are the velocities of the fluid and the particles, respectively.

[0140] Fluids usually flow in a Poiseuille flow state in microchannels. When particles move in the microchannels, they are subject to a force caused by a velocity gradient, namely, shear gradient lift, which is expressed as:

[0141]

[0142] f LS is the dimensionless lift coefficient. In addition to the shear gradient lift, the particles are also affected by the channel wall. There are generally two types of lift caused by the wall. One is the Saffman lift. The existence of the wall will produce a velocity gradient. When the particle is close to the wall, the wall will give the particle additional resistance. The lift generated by the sliding shear between the particle and the wall is the Saffman lift, and the expression is:

[0143]

[0144] Among them, K = 81.2, γ is the fluid velocity gradient shear rate. The other is the wall lift. For particles far from the wall, they will move toward the wall under the action of the shear gradient lift. The wall will produce a wall lift that inhibits the movement of particles toward the wall. The expression is:

[0145]

[0146] Where fLW is the lift coefficient. In the DLD chip, the particles and the wall mainly interact with each other by sliding shear, so the Saffman lift is used to characterize the interaction between the particles and the wall.

[0147] We used finite element software to establish a particle trajectory tracking model to verify the particle motion behavior under the synergistic influence of fluid flow and electric field

[0148] 2-4. Results and Discussion

[0149] When the inlet and outlet of the conventional micro-column 135 array micro-channel (DLD) are energized, an inhomogeneous electric field is generated between the insulating micro-columns 135 perpendicular to the mainstream direction. The electric field intensity distribution is as follows: Fig.16 The electric field intensity between the micropillars 135 perpendicular to the mainstream direction is symmetrically distributed, the closer the electric field intensity between the micropillars 135 is to the wall of the micropillar 135, the smaller it is to the center of the channel, and the electric field intensity distribution upstream and downstream of the micropillar 135 is just the opposite, the closer to the wall of the micropillar 135, the smaller it is, and the farther away from the wall of the micropillar 135, the larger it is.

[0150] Then, we released particles of 5 μm and 8.6 μm along the center line after focusing in the above electric field and flow field. According to the critical diameter empirical calculation formula of Inglis et al., we calculated that the critical diameter in the absence of electric field was 10 μm (reference: Inglis DW, Davis JA, Austin RH, et al. Critical particlesize for fractionation by deterministic lateral displacement [J]. Lab on a Chip, 2006, 6 (5): 655).

[0151] The above situation is numerically simulated using the simulation model after model verification. The simulation results are as follows: Fig.17As shown in the figure, in the absence of an electric field, the red streamline in the figure is the motion trajectory of the 8.6μm particle, and the blue streamline is the motion trajectory of the 5μm particle. The particle sizes of both particles are smaller than the critical diameter of 10um, so when the microchannel is not powered, they both flow in a zigzag shape. When the microchannel is connected to an electric field, the 8.6μm particle begins to "collision" and the 5μm particle moves in a zigzag shape. This phenomenon shows that the electrophoretic force provided by applying an electric field in the micropillar 135 array can reduce the critical diameter. This phenomenon can be used to optimize the structure of the micropillar 135 array and / or synergistically sort particles.

[0152] In addition, we also observed the trajectory behavior of 6μm particles in micro-pillar 135 array microchannels with different structures to verify the accuracy of our above conclusions. The numerical simulation results are shown in Fig.18 As shown: It can be found that as the voltage increases, the critical diameter decreases. When the voltage is 5V, the critical diameter is smaller and closer than that in the case of no electric field. When the voltage is 10V, the critical diameter is significantly reduced, indicating that the dielectrophoretic force can effectively reduce the critical diameter of the DLD device.

[0153] Voltage is applied to the inlet and outlet of the DLD array chip to generate an inhomogeneous electric field through the insulating micropillars 135, so that the particles in the DLD chip are affected by the dielectrophoretic force. Our research shows that the diameter of the DLD chip after integrating iDEP can be reduced, and the critical diameter of iDEP-DLD decreases with the increase of voltage and increases with the increase of flow rate.

[0154] This phenomenon can be used to optimize the structure of traditional DLD chips, and a structural design of DLD chips with integrated iDEP is proposed, which brings two beneficial effects:

[0155] 1. In the design of DLD chips, we can use the characteristic that the critical diameter of the DLD structure becomes smaller after the electric field is added. Combined with the influence of the gap between the micro-pillars 135 of the DLD chip itself on the critical diameter, we can increase the gap between the micro-pillars 135 of the DLD chip without changing the original critical diameter, thereby improving or even solving the shortcoming that traditional DLD chips are easily blocked.

[0156] 2. In the actual process of sorting cells or particles, when the microchannel of the microcolumn 135 structure has been processed, there is a certain critical diameter. Therefore, the traditional DLD chip cannot flexibly adjust the critical diameter of the microcolumn 135 array according to the actual sorting conditions. The DLD chip integrated with iDEP proposed by us can change the critical diameter of the microcolumn 135 array according to the actual sorting conditions by adjusting the applied voltage and flow rate, so as to achieve multiple uses of one core.

[0157] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes, characterized in that: The invention comprises a fixed plate (1), wherein a first sorting module (11) for separating cells and cell exosomes, an enrichment module (12) for enriching cell exosomes, and a second sorting module (13) for sorting normal blood cells and circulating tumor cells are arranged in the fixed plate (1), the inlet of the enrichment module (12) is connected to the exosome outlet of the first sorting module (11), the inlet of the second sorting module (13) is connected to the cell outlet of the first sorting module (11), and 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).

2. The microfluidic device for sorting circulating tumor cells and enriching cell exosomes according to claim 1, characterized in that: The enrichment module (12) comprises a first channel (121) horizontally arranged in a fixed plate (1), an enrichment chamber (122) arranged at the bottom of the first channel (121) and having a circular cross-section, and a laser (123) located directly above the enrichment chamber (122) for heating a suspension in the enrichment chamber (122); a heating area of ​​the laser (123) is cylindrical and arranged in the center of the enrichment chamber (122); the outer diameter of the heating area is smaller than the outer diameter of the enrichment chamber (122); and a structure for increasing the synergistic effect of thermophoresis and thermal convection is provided at the bottom of the enrichment chamber (122).

3. The microfluidic device for sorting circulating tumor cells and enriching cell exosomes according to claim 2, characterized in that: The structure for increasing the synergistic effect of thermophoresis and thermal convection is a top cone (124) disposed at the bottom of the enrichment chamber (122); the top cone (124) is arranged in the center of the enrichment chamber (122); and the generatrix of the top cone (124) is an arc.

4. The microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes according to claim 2 or 3, characterized in that: The bottom of the first channel (121) has a circular hole (125) which is arranged to pass downward through the fixing plate (1), and an enrichment seat (126) is sealed in the circular hole (125), and the enrichment chamber (122) is centrally arranged on the enrichment seat (126).

5. The microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes according to claim 4, characterized in that: A base plate (2) is provided below the fixing plate (1), 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) matching with the limiting boss (21) is provided on the lower side of the fixing plate (1), and the circular hole (125) is provided in the limiting cavity (127).

6. The microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes according to claim 1, characterized in that: The first sorting module (11) comprises a curved microchannel (111) arranged in a fixed plate (1) and a rectangular cavity (112) arranged on the inner side of the curve of the curved microchannel (111); the rectangular cavities (112) are multiple and are evenly arranged along the length direction of the curved microchannel (111); the multiple rectangular cavities (112) are connected to the curved microchannel (111); the cell outlet of the first sorting module (11) is connected to the inner side of the curve of the curved microchannel (111); and the exosome outlet of the first sorting module (11) is connected to the outer side of the curve of the curved microchannel (111).

7. The microfluidic device for sorting circulating tumor cells and enriching cell exosomes according to claim 1, characterized in that: The second sorting module (13) comprises a sorting chamber (131) arranged in the fixed plate (1), a second channel (132) connected to the sorting chamber (131), a normal blood cell outlet (133) and a circulating tumor cell outlet (134); the inlet end of the second channel (132) is connected to the cell outlet of the first sorting module (11); a plurality of array-distributed microcolumns (135) are vertically arranged in the sorting chamber (131); the plurality of microcolumns (135) are obliquely arranged 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.

8. The microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes according to claim 7, characterized in that: The sorting chamber (131) is provided with two electrophoresis plates (136) arranged opposite to each other, and a flow channel (137) is formed between the two electrophoresis plates (136). There are two circulating tumor cell outlets (134) arranged close to the inner sides of different electrophoresis plates (136), and 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 pole of the AC power supply (139) is connected to the second normal blood cell outlet (138), and the positive pole of the AC power supply (139) is connected to the second channel (132).

9. The microfluidic device capable of sorting circulating tumor cells and enriching cell exosomes according to claim 8, characterized in that: The sheath fluid input module (14) includes a plurality of sheath fluid channels (141) connected to the second channel (132), and the positive pole of the AC power source (139) is connected to one of the sheath fluid channels (141).

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