CN111748467B

CN111748467B - Circulating tumor cell detection and sorting device and manufacturing method thereof

Info

Publication number: CN111748467B
Application number: CN202010515908.1A
Authority: CN
Inventors: 倪中华; 项楠; 朱树
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2022-07-12
Anticipated expiration: 2040-06-09
Also published as: CN111748467A

Abstract

The invention discloses a circulating tumor cell detecting and sorting device and a manufacturing method thereof.A sorting layer of the device is respectively communicated with an upper detection layer and a lower detection layer through an upper connecting layer and a lower connecting layer, and the lower detection layer is also communicated with a deformation layer and a control layer; the sample liquid containing leucocytes and circulating tumor cells flows into the sorting layer through the upper connecting layer, and is influenced by the gas pressure of the metal electrodes and the control layer on the upper detection layer and the lower detection layer in the sorting layer, when the circulating tumor cells are detected, the circulating tumor cells are sorted to the collecting pipe for collecting the circulating tumor cells through the upper connecting layer and the upper detection layer, and when the leucocytes are detected, the leucocytes are sorted to the collecting pipe for collecting the leucocytes through the lower connecting layer, the lower detection layer, the deformation layer and the control layer in sequence. The invention can realize the non-labeled separation of the circulating tumor cells and the white blood cells by controlling the flow resistance while detecting the circulating tumor cells and the white blood cells, and has wide application value in the fields of cancer diagnosis, treatment and the like.

Description

Circulating tumor cell detection and sorting device and manufacturing method thereof

Technical Field

The invention relates to a cell separation device, in particular to a circulating tumor cell detection and sorting device and a manufacturing method thereof.

Background

Cancer, also known as malignant tumor, is a disease caused by the malfunction of the mechanism controlling cell growth and proliferation. Malignant tumors grow rapidly and can destroy normal tissues and organs of the human body, and finally cause death of the patient. With the change of living habits and living environments of people, the current situation of cancer is more severe and becomes the most major problem affecting the public health in the world. Modern medicine finds that in the early stages of cancer recurrence and metastasis, tumor cells are shed from primary tumor foci into peripheral blood. Tumor cells (also called circulating tumor cells) in peripheral blood are often used for predicting survival of cancer patients, and can also be used for guiding cancer diagnosis and prognosis evaluation, so that a thought is provided for developing anti-cancer drugs. Therefore, the ability to rapidly and efficiently obtain tumor cells from peripheral blood would be of great significance for cancer diagnosis and treatment.

However, the circulating tumor cell sorting detection device represented by the CellSearch system of qiangsheng corporation in the united states often uses immunomagnetic bead labeling and fluorescent staining to capture and detect tumor cells. Wherein, the captured circulating tumor cells lose biological activity and cannot be used for subsequent clinical diagnosis, drug resistance detection and the like. In addition, such devices based on immunomagnetic bead labeling and fluorescent staining for capturing and detecting circulating tumor cells are often very expensive to use due to the high price of magnetic beads and fluorescent stains. Therefore, the development of a tumor cell sorting device using a non-biochemical labeling method is of great value for the early diagnosis, prognosis evaluation and development of anticancer drugs of cancer.

Disclosure of Invention

The purpose of the invention is as follows: one object of the present invention is to provide a device for detecting and sorting circulating tumor cells that can achieve non-biochemical markers.

Another object of the present invention is to provide a method for making the above-described device.

The technical scheme is as follows: the invention discloses a circulating tumor cell detecting and sorting device, which sequentially comprises an upper detecting layer, an upper connecting layer, a sorting layer, a lower connecting layer, a lower detecting layer, a deformation layer and a control layer from top to bottom, wherein the upper detecting layer and the lower detecting layer are fixedly connected through positioning mounting holes, metal electrodes are arranged on the upper detecting layer and the lower detecting layer, the upper detecting layer is communicated with the sorting layer through the upper connecting layer, the lower detecting layer is communicated with the sorting layer through the lower connecting layer, and the lower detecting layer is also communicated with the deformation layer and the control layer; the sample liquid containing the leucocytes and the circulating tumor cells flows into the sorting layer through the upper connecting layer, and is influenced by the gas pressure of the metal electrodes and the control layer on the upper detection layer and the lower detection layer in the sorting layer.

Preferably, the upper detection layer is provided with a first sample solution inlet, a first circulating tumor cell outlet and a first metal electrode channel, wherein one side of the tail end of the first metal electrode channel, which is connected with the upper connection layer, is provided with a hole, and a metal electrode is inserted into the first metal electrode channel and sealed by using a sealant.

Preferably, the upper connecting layer is provided with a second sample liquid inlet, a second circulating tumor cell outlet and a first metal electrode through hole, wherein the second sample liquid inlet is communicated with the first sample liquid inlet on the upper detection layer, the second circulating tumor cell outlet is communicated with the first circulating tumor cell outlet, and the first metal electrode through hole is communicated with the opening at the tail end of the first metal electrode channel on the upper detection layer.

Preferably, a third sample liquid inlet, a sinusoidal focusing flow channel, a straight flow channel, a first leukocyte outlet, a circulating tumor cell flow channel and a third circulating tumor cell outlet are arranged on the sorting layer, wherein the third sample liquid inlet is communicated with the sinusoidal focusing flow channel, the sinusoidal focusing flow channel is communicated with the straight flow channel, the straight flow channel is divided into two paths, one path is communicated with the first leukocyte flow channel, the tail end of the first leukocyte flow channel is communicated with the first leukocyte outlet, the other path is communicated with the circulating tumor cell flow channel, and the tail end of the circulating tumor cell flow channel is communicated with the third circulating tumor cell outlet; the third sample liquid inlet is communicated with the first sample liquid inlet on the upper detection layer through the second sample liquid inlet on the upper connecting layer, the third circulating tumor cell outlet is communicated with the first circulating tumor cell outlet on the upper detection layer through the second circulating tumor cell outlet on the upper connecting layer, and a through hole which is completely penetrated is formed in the direct current channel close to the sinusoidal focusing flow channel and is respectively communicated with the first metal electrode through hole on the upper connecting layer and the second metal electrode through hole on the lower connecting layer through the through hole.

Preferably, the lower connecting layer is provided with a second metal electrode through hole and a second leukocyte outlet, wherein the second metal electrode through hole is communicated with the direct current channel on the sorting layer, and the second leukocyte outlet is communicated with the first leukocyte outlet.

Preferably, the lower detection layer is provided with a second metal electrode channel, a leukocyte inlet, a second leukocyte channel and a third leukocyte outlet, wherein two ends of the second leukocyte channel are respectively communicated with the leukocyte inlet and the third leukocyte outlet, the leukocyte inlet is communicated with the second leukocyte outlet of the lower connecting layer, the tail end of the second metal electrode channel is communicated with the second metal electrode through hole of the lower connecting layer, a metal electrode is inserted into the second metal electrode channel, and the tail end of the second metal electrode channel is provided with a hole at one side connected with the lower connecting layer.

Preferably, the deformation layer is provided with a fourth leukocyte outlet, wherein the deformation layer directly seals the leukocyte inlet of the lower detection layer and one side of the second leukocyte flow channel, and the fourth leukocyte outlet is communicated with the third leukocyte outlet of the lower detection layer.

Preferably, a control gas inlet, a control gas channel, a fifth leukocyte outlet and a control gas outlet are arranged on the control layer, wherein the deformation layer seals one side of the control gas channel of the control layer together, two ends of the control gas channel are respectively communicated with the control gas inlet and the control gas outlet, the fifth leukocyte outlet is communicated with a fourth leukocyte outlet of the deformation layer, and the control gas channel, the deformation layer above and a second leukocyte flow channel of the lower detection layer jointly form a flow resistance adjusting structure.

Preferably, the upper detection layer, the sorting layer, the lower detection layer, the deformation layer and the control layer are one or more of polydimethylsiloxane PDMS, silica gel, plastic and glass, and the upper connection layer and the lower connection layer are double-sided adhesive tapes.

The preparation method of the circulating tumor cell detecting and sorting device comprises the following steps:

(S1) selecting PVC plastic to prepare an upper detection layer and a lower detection layer, and selecting a silica gel material to prepare a separation layer;

the upper detection layer, the sorting layer and the lower detection layer are of a three-piece structure, the middle is silica gel, and the upper and lower pieces are PVC plastic;

when the detection layer is manufactured, two PVC plastic substrates and one silica gel material substrate are selected, then an upper detection layer and a required structure are respectively etched by a laser, and then packaging is completed through a plasma bonding technology to form the detection layer;

secondly, selecting two PVC plastic substrates and a silica gel material substrate, then respectively etching structures required by a sorting layer by using a laser, and then completing encapsulation by using a plasma bonding technology to form the sorting layer;

then, selecting two PVC plastic substrates and a silica gel material substrate, then respectively etching a structure required by the lower detection layer by using a laser, and completing encapsulation by using a plasma bonding technology to form the lower detection layer;

(S2) selecting a double-faced adhesive material to prepare an upper connecting layer and a lower connecting layer;

when in manufacturing, the structures needed by the upper connecting layer and the lower connecting layer are carved on the double-faced adhesive material with the PVC substrate covered on the selected surface by a laser, and the PVC substrate faces downwards when in laser processing; the stacking of the upper detection layer, the sorting layer and the lower detection layer is realized through the double-sided adhesion of the upper connecting layer and the lower connecting layer;

(S3) preparing a deformation layer by selecting silica gel;

during manufacturing, structures required by the deformation layer are respectively etched on the selected PVC substrate and the selected silica gel substrate by using a laser;

(S4) selecting PVC plastic and silica gel materials to prepare a control layer;

when the control layer is manufactured, the control layer is of a two-piece structure, the first piece is made of PVC plastic, the second piece is made of a silica gel material, structures required by the control layer are respectively carved on a selected PVC substrate and a selected silica gel substrate by a laser, and the two pieces are bonded together by a plasma bonding technology;

(S5) during assembly, firstly, the upper detection layer is adhered to the upper connection layer, then the PVC protective film on the double-faced adhesive material is torn off, and the PVC protective film is adhered to the sorting layer; the other side of the sorting layer is adhered to the lower connecting layer, and then the PVC protective film on the double-faced adhesive material is torn off and is adhered to the lower detection layer; and bonding the two sheets together by the lower detection layer and the deformation layer through a plasma bonding technology, and then bonding the other surface of the deformation layer and the control layer together through the plasma bonding technology.

Description of the principle:

the first sample liquid inlet and the first circulating tumor cell outlet of the upper detection layer are respectively connected with an injector filled with sample liquid and a collecting pipe for collecting circulating tumor cells, and the control gas inlet, the control gas outlet and the fifth leucocyte outlet of the control layer are respectively connected with the inlet of the air pressure regulating device, the outlet of the air pressure regulating device and the collecting pipe for collecting leucocytes. When the sample liquid containing the white blood cells and the circulating tumor cells is used, the sample liquid sequentially flows into the sinusoidal focusing flow channel of the sorting layer through the first sample liquid inlet, the second sample liquid inlet and the third sample liquid inlet of the upper detection layer and the sorting layer so as to realize the inertial focusing of the white blood cells and the circulating tumor cells; the principle is as follows: when the fluid flows in the bent flow channel, the white blood cells and the circulating tumor cells are under the action of inertial lift force and Dean drag force, and the white blood cells and the circulating tumor cells are focused into a single row; the metal electrodes are inserted into the metal electrode channels arranged on the upper detection layer and the lower detection layer and are connected with the direct current channel of the sorting layer through the metal electrode through holes of the upper connection layer and the lower connection layer, so that an electric field can be generated in a region where the metal electrode through holes are connected with the direct current channel when the metal electrodes are electrified, and when white blood cells and circulating tumor cells flow through the region, which cells are the white blood cells and the circulating tumor cells can be distinguished; the principle is as follows: when white blood cells or circulating tumor cells flow through the electric field area, the electric field is disturbed, and electric field disturbances generated by different types of cells are different; when the circulating tumor cells are detected, the gas pressure of a control gas channel of the control layer is increased, and the deformation layer is extruded and deformed, so that the flow resistance of a white blood cell flow channel of the lower detection layer is increased, and the flow resistance of a white blood cell flow channel of the sorting layer is increased, so that the circulating tumor cells only can flow into the circulating tumor cell channel with smaller flow resistance, and are finally sorted into a collecting pipe for collecting the circulating tumor cells; when the white blood cells flow through the electric field area and are detected, the control gas channel of the control layer is not required to be increased in gas pressure, so that the flow resistance of the white blood cell flow channel of the separation layer and the circulating tumor cell flow channel cannot be changed, the white blood cells only can flow into the white blood cell flow channel with smaller flow resistance, and the white blood cells are finally separated to the collecting pipe for collecting the white blood cells.

Has the advantages that: compared with the prior art, the device can realize non-labeled sorting of the circulating tumor cells and the white blood cells by controlling the flow resistance while detecting the circulating tumor cells and the white blood cells, can detect and sort the living circulating tumor cells from the sample liquid containing the white blood cells and the circulating tumor cells, has wide application value in the fields of cancer diagnosis, treatment and the like, and provides a quick and effective mode for the subsequent clinical diagnosis and other applications of cancer.

Drawings

FIG. 1 is an exploded view of the assembly of the apparatus of the present invention;

FIG. 2 is a schematic view of the upper detection layer structure;

FIG. 3 is a schematic view of the upper connection layer structure;

FIG. 4 is a schematic view of a sorting layer structure;

FIG. 5 is a schematic view of a lower connection layer structure;

FIG. 6 is a schematic view of the lower detection layer structure;

FIG. 7 is a schematic diagram of a morphable layer structure;

FIG. 8 is a schematic diagram of a control layer structure;

FIG. 9 is a schematic sectional view of a flow resistance adjusting structure;

FIG. 10 is a schematic cross-sectional view of a flow resistance adjusting structure after deformation;

FIG. 11 is a top view of the stacked tumor cell detection and sorting device;

FIG. 12 shows the result of measurement of an electrical impedance detection signal of a cell.

Detailed Description

For a further understanding of the present invention, reference will now be made in detail to the embodiments illustrated in the drawings. The following are only preferred embodiments of the present invention, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

As shown in figure 1, the circulating tumor cell detecting and sorting device is formed by stacking an upper detecting layer 1, an upper connecting layer 2, a sorting layer 3, a lower connecting layer 4, a lower detecting layer 5, a deformation layer 6 and a control layer 7 from top to bottom, and positioning mounting holes are uniformly formed in the edge position of each layer of structure.

As shown in fig. 2, the upper detection layer 1 is provided with a first sample solution inlet 11, a first circulating tumor cell outlet 12, and a first metal electrode channel 13; wherein, a metal electrode is inserted into the first metal electrode channel 13 and sealed by sealant; the metal electrode is a silver electrode, and the end of the first metal electrode channel is provided with a hole at one side connected with the upper connecting layer.

As shown in fig. 3, the upper connection layer 2 is provided with a second sample solution inlet 21, a second circulating tumor cell outlet 22, and a first metal electrode through hole 23; the second sample solution inlet 21 and the second circulating tumor cell outlet 22 are respectively communicated with the first sample solution inlet 11 and the first circulating tumor cell outlet 12 on the upper detection layer 1, and the first metal electrode through hole 23 is communicated with the opening at the tail end of the first metal electrode channel 13 on the upper detection layer 1.

As shown in fig. 4, the sorting layer 3 is provided with a third sample solution inlet 31, a sinusoidal focusing flow channel 32, a straight flow channel 33, a first leukocyte flow channel 34, a first leukocyte outlet 35, a circulating tumor cell flow channel 36, and a third circulating tumor cell outlet 37; the third sample liquid inlet 31 is communicated with the sinusoidal focusing flow channel 32, the sinusoidal focusing flow channel 32 is communicated with the direct flow channel 33, the direct flow channel 33 is divided into two paths, one path is communicated with the first leukocyte flow channel 34, the tail end of the first leukocyte flow channel 34 is communicated with the first leukocyte outlet 35, the other path is communicated with the circulating tumor cell flow channel 36, and the tail end of the circulating tumor cell flow channel 36 is communicated with the third circulating tumor cell outlet 37; the third sample solution inlet 31 is communicated with the first sample solution inlet 11 on the upper detection layer 1 through the second sample solution inlet 21 on the upper connecting layer 2, the third circulating tumor cell outlet 37 is communicated with the first circulating tumor cell outlet 12 on the upper detection layer 1 through the second circulating tumor cell outlet 22 on the upper connecting layer 2, and a through hole which completely penetrates is formed in the straight flow channel 33 close to the sinusoidal focusing flow channel 32 and is respectively communicated with the first metal electrode through hole 23 on the upper connecting layer 2 and the second metal electrode through hole 41 on the lower connecting layer 4 through the through hole. The sinusoidal focusing flow channel is not contacted with the first metal electrode through hole of the upper connecting layer and the second metal electrode through hole of the lower connecting layer.

As shown in fig. 5, the lower connection layer 4 is provided with a second metal electrode through hole 41 and a second leukocyte outlet 42; the second metal electrode through hole 41 and the second leukocyte outlet 42 are respectively communicated with the straight flow channel 33 and the first leukocyte outlet 35 on the sorting layer 3.

As shown in fig. 6, the lower detection layer 5 is provided with a second metal electrode channel 51, a leukocyte inlet 52, a second leukocyte flow channel 53, and a third leukocyte outlet 54; wherein, two ends of the second leukocyte flow channel 53 are respectively communicated with a leukocyte inlet 52 and a third leukocyte outlet 54, the leukocyte inlet 52 is connected with the second leukocyte outlet 42 of the lower connecting layer 4, the tail end of the second metal electrode channel 51 is connected with the second metal electrode through hole 41 of the lower connecting layer 4, and a metal electrode is inserted into the second metal electrode channel 51. The metal electrode is a silver electrode, and the tail end of the second metal electrode channel is provided with a hole at one side connected with the lower connecting layer.

As shown in fig. 7, the shape-changing layer 6 is provided with a fourth leukocyte outlet 61; the deformable layer 6 directly seals the white blood cell inlet 52 of the lower detection layer 5 and one side of the second white blood cell channel 53, and the fourth white blood cell outlet 61 is communicated with the third white blood cell outlet 54 of the lower detection layer 5. The shape of the deformation layer is set to be a shape formed by splicing a trapezoid and a rectangle, and the fourth white blood cell outlet of the deformation layer is a hole which is completely penetrated.

As shown in fig. 8, the control layer 7 is provided with a control gas inlet 71, a control gas channel 72, a fifth leukocyte outlet 73, and a control gas outlet 74; the shape changing layer 6 seals one side of the control gas channel 72 of the control layer 7, two ends of the control gas channel 72 are respectively communicated with the control gas inlet 71 and the control gas outlet 74, the fifth leukocyte outlet 73 is connected with the fourth leukocyte outlet 61 of the shape changing layer 6, and the control gas channel 72, the shape changing layer 6 above and the second leukocyte flow channel 53 of the lower detection layer 5 jointly form a flow resistance adjusting structure. The shape of the control layer is set to be a shape of splicing a trapezoid and a rectangle, and the control gas inlet and the control gas outlet of the control layer are holes with openings on one side. The direction of the control gas channel intersects with the direction of the second leukocyte flow channel of the lower detection layer from the top view.

The upper detection layer, the sorting layer, the lower detection layer, the deformation layer and the control layer are one or more of polydimethylsiloxane PDMS, silica gel, plastic and glass, and the upper connection layer and the lower connection layer are double-sided adhesive tapes.

As shown in fig. 9, which is a sectional view of the flow resistance adjusting structure, the flow channel direction of the second leukocyte flow channel 53 of the lower detection layer 5 intersects with the channel direction of the control gas channel 72 on the control layer 7. When the control gas is not introduced into the control gas channel 72, the deformable layer 6 is not deformed, and the second leukocyte flow channel 53 is not pressed.

As shown in the schematic cross-sectional view of the flow resistance adjusting structure after deformation in fig. 10, when the control gas is introduced into the control gas channel 72 on the control layer 7, the deformable layer 6 is squeezed, and further the channel of the second leukocyte flow channel 53 of the lower detection layer 5 is squeezed.

As shown in fig. 11, in the plan view of the stacked circulating tumor cell detecting and sorting apparatus, the upper and lower detecting

layers

1 and 5 are communicated with the straight flow channels 33 of the sorting layer 3 at positions near the sinusoidal focusing flow channels 32 of the sorting layer 3, and the direction of the control gas channel 72 of the control layer 7 and the direction of the second leukocyte flow channel 53 of the lower detecting layer 5 intersect with each other in the plan view.

As shown in fig. 1 to 11, the device for detecting and sorting circulating tumor cells of the present invention has an overall structure formed by stacking an upper detection layer 1, an upper connection layer 2, a sorting layer 3, a lower connection layer 4, a lower detection layer 5, a deformation layer 6, and a control layer 7 from top to bottom. The first sample liquid inlet 11 and the first circulating tumor cell outlet 12 of the upper detection layer 1 are respectively connected with an injector filled with sample liquid and a collecting pipe for collecting circulating tumor cells, and the control gas inlet 71, the fifth leukocyte outlet 73 and the control gas outlet 74 of the control layer 7 are respectively connected with an outlet of an air pressure regulating device, a collecting pipe for collecting leukocytes and an outlet of an air pressure regulating device. The control gas channel 72 of the control layer 7, the upper deformation layer 5 and the second leukocyte flow channel 53 of the lower detection layer 5 together form a flow resistance adjusting structure. When in use, a sample solution containing white blood cells and circulating tumor cells sequentially flows through the first sample solution inlet 11 of the upper detection layer 1, the second sample solution inlet 21 of the upper connecting layer 2 and the third sample solution inlet 31 of the sorting layer 3 to enter the sinusoidal focusing flow channel 32; when the cell sample flows to the connection part of the direct current channel 33 and the first metal electrode through hole 23 and the second metal electrode through hole 41, the cell is detected by the electric field with specific frequency and amplitude; the cell sample liquid flow causes the disturbance of the electric field when passing through the electric field applied by the metal electrodes of the upper detection layer and the lower detection layer. As shown in fig. 12, the perturbation of the electric field caused by the circulating tumor cells passing through this field is greater than that of the white blood cells. When the circulating tumor cells pass through the electric field area, the induced electric field disturbance is large, so that the gas pressure is increased to the control gas channel 72 of the control layer 7, and the deformation layer 6 is extruded and deformed. Therefore, the flow resistance of the second leukocyte flow channel 53 of the lower detection layer 5 is increased, the flow resistance of the first leukocyte flow channel 34 of the sorting layer 3 is increased, and the circulating tumor cells can only flow into the circulating tumor cell channel 36 with smaller flow resistance, and finally are sorted to the collection tube for collecting the circulating tumor cells. When the white blood cells flow through the electric field area, the disturbance of the electric field caused by the white blood cells is small, and the control gas channel 72 of the control layer 7 does not need to increase the gas pressure, so that the flow resistance of the first white blood cell flow channel 34 of the sorting layer 3 and the circulating tumor cell flow channel 36 is not changed, and the white blood cells can only flow into the first white blood cell flow channel 34 with small flow resistance, and finally are sorted into the collection tube for collecting the white blood cells. The flow resistance of the first leukocyte flow channel 34 and the circulating tumor cell flow channel 36 is related to the sectional area of the flow channels, and the larger the sectional area of the flow channels is, the smaller the flow resistance is; as shown in FIG. 4, the first leukocyte flow channel 34 is significantly larger in cross-sectional area than the circulating tumor cell flow channel 36, and therefore has a lower flow resistance.

In this embodiment, the speed of detecting and sorting circulating tumor cells can be increased by paralleling a plurality of circulating tumor cell detecting and sorting devices of the present invention.

In this embodiment, the upper detection layer 1, the sorting layer 3, and the lower detection layer 5 are 3-piece structures, the first upper detection layer 1 and the third lower detection layer 5 are PVC plastic, and the second sorting layer 3 is a silica gel material. During manufacturing, the required structures are respectively carved on the selected PVC substrate and the selected silica gel substrate by a laser, and then packaging is completed by a plasma bonding technology. The technology has the advantages of short processing time (<1 min/piece), high processing precision (deviation about 5 mu m), low manufacturing cost and strong flexibility. The upper connecting layer 2 and the lower connecting layer 4 are made of double-sided adhesive materials. When in manufacturing, the required structure is carved on the double-faced adhesive material with the PVC substrate covered on the selected surface by a laser, and the PVC substrate faces downwards when in laser processing. The stacking of the upper detection layer 1, the sorting layer 3 and the lower detection layer 5 is realized by the double-sided adhesion of the upper connecting layer 2 and the lower connecting layer 4. The deformation layer 6 is of a monolithic structure and is made of silicone. During manufacture, a laser is used for carving a required structure on the selected silica gel substrate. The control layer 7 is 2 structures, and first piece is PVC plastics, and the second piece is the silica gel material. When in manufacturing, the required structures are respectively carved on the selected PVC substrate and the selected silica gel substrate by a laser, and then the two pieces are bonded together by a plasma bonding technology. During assembly, the upper detection layer 1 and the upper connecting layer 2 are firstly adhered together, then the PVC protective film on the double-faced adhesive material is torn off, and the PVC protective film and the sorting layer 3 are adhered together. The other side of the sorting layer 3 is adhered with the lower connecting layer 4, then the PVC protective film on the double-faced adhesive material is torn off, and the PVC protective film is adhered with the lower detection layer 5. The lower detection layer 5 and the deformation layer 6 are bonded together by a plasma bonding technology, and then the other side of the deformation layer 6 is bonded together with the control layer by the plasma bonding technology.

Claims

1. A circulating tumor cell detecting and sorting device is characterized by comprising an upper detecting layer (1), an upper connecting layer (2), a sorting layer (3), a lower connecting layer (4), a lower detecting layer (5), a deformation layer (6) and a control layer (7) from top to bottom in sequence, wherein the layers are fixedly connected through positioning mounting holes, metal electrodes are arranged on the upper detecting layer (1) and the lower detecting layer (5), the upper detecting layer (1) is communicated with the sorting layer (3) through the upper connecting layer (2), the lower detecting layer (5) is communicated with the sorting layer (3) through the lower connecting layer (4), and the lower detecting layer (5) is also communicated with the deformation layer (6) and the control layer (7); a sample liquid containing white blood cells and circulating tumor cells flows into a sorting layer through an upper connecting layer (2), and is influenced by gas pressure of metal electrodes and a control layer on an upper detection layer (1) and a lower detection layer (5) in the sorting layer, when the circulating tumor cells are detected, the circulating tumor cells are sorted to a collecting pipe for collecting the circulating tumor cells through the upper connecting layer (2) and the upper detection layer (1), and when the white blood cells are detected, the white blood cells are sorted to the collecting pipe for collecting the white blood cells through a lower connecting layer (4), the lower detection layer (5), a deformation layer (6) and the control layer (7) in sequence.

2. The device for detecting and sorting the circulating tumor cells according to claim 1, wherein the upper detecting layer (1) is provided with a first sample solution inlet (11), a first circulating tumor cell outlet (12) and a first metal electrode channel (13), wherein the end of the first metal electrode channel (13) is provided with an opening at one side connected with the upper connecting layer, and a metal electrode is inserted into the first metal electrode channel (13) and sealed by a sealant.

3. The device for detecting and sorting the circulating tumor cells according to claim 1, wherein the upper connecting layer (2) is provided with a second sample solution inlet (21), a second circulating tumor cell outlet (22) and a first metal electrode through hole (23), wherein the second sample solution inlet (21) is communicated with the first sample solution inlet (11) on the upper detecting layer (1), the second circulating tumor cell outlet (22) is communicated with the first circulating tumor cell outlet (12), and the first metal electrode through hole (23) is communicated with the opening at the end of the first metal electrode channel (13) on the upper detecting layer (1).

4. The device for detecting and sorting circulating tumor cells according to claim 1, wherein the sorting layer (3) is provided with a third sample solution inlet (31), a sinusoidal focusing flow channel (32), a direct flow channel (33), a first leukocyte flow channel (34), a first leukocyte outlet (35), a circulating tumor cell flow channel (36) and a third circulating tumor cell outlet (37), wherein the third sample liquid inlet (31) is communicated with the sine focusing flow channel (32), the sine focusing flow channel (32) is communicated with the straight flow channel (33), the straight flow channel (33) is divided into two paths, one path is communicated with the first leukocyte flow channel (34), the tail end of the first leucocyte flow channel (34) is communicated with the first leucocyte outlet (35), the other path is communicated with the circulating tumor cell flow channel (36), and the tail end of the circulating tumor cell flow channel (36) is communicated with a third circulating tumor cell outlet (37); the third sample liquid inlet (31) is communicated with the first sample liquid inlet (11) on the upper detection layer (1) through the second sample liquid inlet (21) on the upper connecting layer (2), the third circulating tumor cell outlet (37) is communicated with the first circulating tumor cell outlet (12) on the upper detection layer (1) through the second circulating tumor cell outlet (22) on the upper connecting layer (2), a through hole which is completely penetrated is formed in the straight flow channel (33) close to the sine focusing flow channel (32), and the through hole is respectively communicated with the first metal electrode through hole (23) on the upper connecting layer (2) and the second metal electrode through hole (41) on the lower connecting layer (4).

5. The device for detecting and sorting circulating tumor cells according to claim 1, wherein the lower connecting layer (4) is provided with a second metal electrode through hole (41) and a second white blood cell outlet (42), wherein the second metal electrode through hole (41) is communicated with the straight flow channel (33) on the sorting layer (3), and the second white blood cell outlet (42) is communicated with the first white blood cell outlet (35).

6. The circulating tumor cell detecting and sorting apparatus of claim 1, wherein the lower detection layer (5) is provided with a second metal electrode channel (51), a leukocyte inlet (52), a second leukocyte flow channel (53) and a third leukocyte outlet (54), wherein both ends of the second leukocyte flow channel (53) are respectively communicated with the leukocyte inlet (52) and the third leukocyte outlet (54), the leukocyte inlet (52) is communicated with the second leukocyte outlet (42) of the lower connection layer (4), the end of the second metal electrode channel (51) is communicated with the second metal electrode through hole (41) of the lower connection layer (4), the metal electrode is inserted into the second metal electrode channel (51), and the end of the second metal electrode channel (51) is opened at a side connected with the lower connection layer.

7. The circulating tumor cell detecting and sorting apparatus of claim 1, wherein the deformation layer (6) is provided with a fourth leukocyte outlet (61), wherein the deformation layer (6) directly seals the leukocyte inlet (52) of the lower detection layer (5) and one side of the second leukocyte flow channel (53), and the fourth leukocyte outlet (61) is communicated with the third leukocyte outlet (54) of the lower detection layer (5).

8. The device for detecting and sorting circulating tumor cells according to claim 1, wherein the control layer (7) is provided with a control gas inlet (71), a control gas channel (72), a fifth leukocyte outlet (73) and a control gas outlet (74), wherein the deformation layer (6) seals one side of the control gas channel (72) of the control layer (7), two ends of the control gas channel (72) are respectively communicated with the control gas inlet (71) and the control gas outlet (74), the fifth leukocyte outlet (73) is communicated with the fourth leukocyte outlet (61) of the deformation layer (6), and the control gas channel (72) and the second leukocyte flow channel (53) of the upper deformation layer (6) and the lower detection layer (5) jointly form a flow resistance adjusting structure.

9. The device for detecting and sorting circulating tumor cells according to claim 1, wherein the upper detection layer (1), the sorting layer (3), the lower detection layer (5), the deformation layer (6) and the control layer (7) are one or more of polydimethylsiloxane PDMS, silica gel, plastic and glass, and the upper connection layer (2) and the lower connection layer (4) are double-sided adhesive tapes.

10. The method for manufacturing a device for detecting and sorting circulating tumor cells of any one of claims 1 to 9, comprising the steps of:

(S1) preparing an upper detection layer (1), a sorting layer (3) and a lower detection layer (5);

the upper detection layer (1), the sorting layer (3) and the lower detection layer (5) are all of a three-piece structure, the middle is silica gel, and the upper and lower pieces are PVC plastic;

when the detection layer is manufactured, two PVC plastic substrates and one silica gel material substrate are selected, then the upper detection layer (1) and a required structure are respectively etched by a laser, and then the packaging is completed through a plasma bonding technology to form the detection layer (1);

secondly, selecting two PVC plastic substrates and a silica gel material substrate, then respectively engraving a structure required by the sorting layer (3) by using a laser, and then finishing packaging by using a plasma bonding technology to form the sorting layer (3);

then, selecting two PVC plastic substrates and a silica gel material substrate, then respectively etching a structure required by the lower detection layer (5) by using a laser, and then completing encapsulation by using a plasma bonding technology to form the lower detection layer (5);

(S2) preparing an upper connecting layer (2) and a lower connecting layer (4) by selecting a double-sided adhesive material;

when the double-faced adhesive tape is manufactured, the structures required by the upper connecting layer (2) and the lower connecting layer (4) are respectively carved on the two selected double-faced adhesive materials of which the surfaces are covered with the PVC substrate by a laser, and the PVC substrate faces downwards when the double-faced adhesive tape is processed by the laser; the stacking of the upper detection layer (1), the sorting layer (3) and the lower detection layer (5) is realized through the double-sided adhesion of the upper connecting layer (2) and the lower connecting layer (4);

(S3) selecting silica gel to prepare a deformation layer (6);

during manufacturing, the structures required by the deformation layer (6) are respectively carved on the selected silica gel substrate by a laser;

(S4) selecting PVC plastic and silica gel materials to prepare the control layer (7);

when the control layer (7) is manufactured, the control layer is of a two-piece structure, the first piece is made of PVC plastic, the second piece is made of silica gel material, structures needed by the control layer (7) are respectively carved on a selected PVC plastic substrate and a selected silica gel material substrate by a laser, and the two pieces are bonded together by a plasma bonding technology;

(S5) during assembly, firstly, the upper detection layer (1) and the upper connecting layer (2) are adhered together, and then the PVC protective film on the double-sided adhesive material is torn off and is adhered together with the sorting layer (3); the other surface of the sorting layer (3) is adhered to the lower connecting layer (4), and then the PVC protective film on the double-faced adhesive material is torn off and is adhered to the lower detection layer (5); the lower detection layer (5) and the deformation layer (6) are bonded together by a plasma bonding technology, and then the other side of the deformation layer (6) is bonded together with the control layer (7) by the plasma bonding technology.