WO2022036891A1

WO2022036891A1 - Device for sorting and testing cancer cells

Info

Publication number: WO2022036891A1
Application number: PCT/CN2020/128486
Authority: WO
Inventors: 倪中华; 项楠; 易红; 朱树
Original assignee: 东南大学
Priority date: 2020-08-19
Filing date: 2020-11-13
Publication date: 2022-02-24
Also published as: CN112111385B; CN112111385A

Abstract

A device for sorting and testing cancer cells. The sorting and test of cancer cells are integrally completed. The device comprises sequentially from top to bottom: an upper clamp, a sorting unit, a test unit and a lower clamp, wherein the upper clamp is provided with a sample liquid inlet, and the lower clamp is provided with a test liquid outlet; and the sample liquid inlet is communicated with an inlet of the sorting unit, an outlet of the sorting unit is communicated with an inlet of the test unit, and an outlet of the test unit is communicated with the test liquid outlet.

Description

A cancer cell sorting and detection device

technical field

The present invention relates to the technical field of cancer cell detection tools, in particular, to a cancer cell sorting and detection device.

Background technique

Cancer, also known as malignant tumor, is a disease caused by the malfunction of the mechanism that controls the growth and proliferation of cells. Malignant tumors grow rapidly and destroy normal tissues and organs in the human body, eventually causing death of the patient. With the change of people's living habits and living environment, the current situation of cancer is more serious, and it has become the most important problem affecting the public health of the world. Modern medicine has found that in the early stage of cancer recurrence and metastasis, tumor cells will fall off from the original tumor foci and enter the peripheral blood. Tumor cells (also known as circulating tumor cells) in peripheral blood are often used to predict the survival of cancer patients, and can also be used to guide cancer diagnosis and prognosis evaluation, and provide ideas for the development of anticancer drugs. Therefore, the ability to obtain tumor cells from peripheral blood quickly and efficiently will be of great significance for cancer diagnosis and treatment.

However, circulating tumor cell sorting and detection devices represented by Johnson & Johnson's CellSearch system often use immunomagnetic bead labeling and fluorescent staining to capture and detect tumor cells. Among them, the captured circulating tumor cells will lose their biological activity and cannot be used for subsequent clinical diagnosis, drug resistance detection, etc. In addition, due to the expensive price of magnetic beads and fluorescent dyes, such devices based on immunomagnetic labeling and fluorescent staining to capture and detect circulating tumor cells are often expensive to use. Therefore, the development of a tumor cell sorting device using a non-biochemical marker method is of great value for the early diagnosis of cancer, prognosis evaluation and anticancer drug development.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a cancer cell sorting and detection device, which integrates the sorting and detection of cancer cells.

In order to solve the above technical problems, an embodiment of the present invention provides a cancer cell sorting and detection device, which includes an upper clamp, a sorting unit, a detection unit, and a lower clamp in order from top to bottom, and the sorting unit is used to separate the cells from the cell fluid. Cancer cells are sorted out in the middle, and the detection unit is used for electrical impedance detection of cancer cells; the upper fixture is provided with a sample liquid inlet, and the lower fixture is provided with a detection liquid outlet; the sample liquid inlet is connected to the sorting unit. The inlet is communicated, the outlet of the sorting unit is communicated with the inlet of the detection unit, and the outlet of the detection unit is communicated with the outlet of the detection liquid.

As a further improvement of the embodiment of the present invention, the sorting unit includes a sorting chip, the sorting chip includes a chip body, and the chip body is provided with a spiral sorting flow channel, a sorting liquid flow channel and a waste liquid flow Both the sorting liquid flow channel and the waste liquid flow channel are connected with the outlet of the spiral sorting flow channel; the inlet of the spiral sorting flow channel is connected with the sample liquid inlet, and the outlet of the sorting liquid flow channel is connected to the inlet of the sample liquid. The inlet of the detection unit is connected.

As a further improvement of the embodiment of the present invention, the sorting unit further includes a sorting liquid concentration regulator for concentrating the concentration of cancer cells sorted by the sorting chip; the sorting liquid concentration regulator is arranged on the upper fixture Between the separation liquid and the separation chip, the separation liquid concentration regulator is respectively communicated with the outlet of the separation liquid flow channel and the inlet of the detection unit.

As a further improvement of the embodiment of the present invention, the separation liquid concentration adjustment member includes an adjustable concentration chip, and the adjustable concentration chip includes a second gas layer, a second elastic layer, a second gas layer, a second elastic layer, a second gas layer, and a second liquid layer and concentrated layer;

The second gas layer is provided with a second gas flow channel, the second liquid layer is provided with a second liquid flow channel, and the second gas flow channel and the second liquid flow channel are arranged in a cross shape;

The concentrated layer is provided with a spiral concentrated flow channel, a concentrated waste liquid flow channel and a concentrated liquid flow channel, and the concentrated waste liquid flow channel and the concentrated liquid flow channel are all connected with the outlet of the spiral concentrated flow channel; The inlet of the concentrated flow channel is communicated with the outlet of the sorting liquid flow channel; the outlet of the concentrated solution flow channel is communicated with the inlet of the detection unit;

The upper clamp is provided with a second regulating gas inlet, a second regulating gas outlet and a second regulating liquid outlet, the second regulating gas inlet is communicated with the inlet of the second gas flow channel, and the second regulating gas outlet is connected with the second regulating gas outlet. The outlets of the two gas flow channels are connected, the second regulating liquid outlet is connected with the outlet of the second liquid flow channel, and the inlet of the second liquid flow channel is connected with the outlet of the concentrated waste liquid flow channel.

As a further improvement of the embodiment of the present invention, a waste liquid flow regulating member is further provided between the upper clamp and the sorting chip, and the waste liquid flow regulating member is connected with the outlet of the waste liquid flow channel, and is used for regulating the waste liquid flow. The flow resistance of the liquid flow path.

As a further improvement of the embodiment of the present invention, the waste liquid flow regulating member includes a flow regulating valve, and the flow regulating valve includes a first gas layer, a first elastic layer, and a first liquid layer that are stacked in sequence from top to bottom, The first gas layer is provided with a first gas flow channel, the first liquid layer is provided with a first liquid flow channel, and the first gas flow channel and the first liquid flow channel are arranged in a cross shape;

The upper fixture is provided with a first regulating gas inlet, a first regulating gas outlet and a first regulating liquid outlet, the first regulating gas inlet is communicated with the inlet of the first gas flow channel, and the first regulating gas outlet is connected with the first regulating gas outlet. The outlet of a gas flow channel is communicated with, the first adjustment liquid outlet is communicated with the outlet of the first liquid flow channel, and the inlet of the first fluid flow channel is communicated with the outlet of the waste liquid flow channel of the sorting chip.

As a further improvement of the embodiment of the present invention, the detection unit includes a superimposed multi-channel detection chip and a circuit connection board, the multi-channel detection chip is connected to the circuit connection board; the multi-channel detection chip includes superimposed electrical impedances The detection layer and the flow channel layer, the electrical impedance detection layer is connected with the circuit connection board, and the flow channel layer is located between the electrical impedance detection layer and the circuit connection board;

The flow channel layer is provided with an evenly divided flow channel, n parallelly arranged detection flow channels and a collection flow channel. The inlet of the detection flow channel is connected, the outlet of the n detection flow channels is connected with the inlet of the collection flow channel, and the outlet of the collection flow channel is connected with the detection liquid outlet of the lower fixture; n represents an integer greater than or equal to 2;

The electrical impedance detection layer is provided with an inlet excitation electrode, n inlet response electrodes, an outlet excitation electrode, n outlet right response electrodes and n outlet left response electrodes; the inlet excitation electrode corresponds to the inlet of the detection flow channel, and the outlet The excitation electrodes correspond to the outlets of the detection channels; the n inlet response electrodes are arranged at intervals along the extension direction of the inlet excitation electrodes, and correspond to the n detection channels respectively; the n outlet right response electrodes correspond to the n The outlet left response electrodes are symmetrically arranged on both sides of the outlet excitation electrode, and are arranged at intervals along the extension direction of the outlet excitation electrode, respectively corresponding to the n detection flow channels one-to-one.

As a further improvement of the embodiment of the present invention, the circuit connection board is provided with inlet excitation electrode connection points, n inlet response electrode connection points, outlet excitation electrode connection points, n outlet right response electrode connection points, and n outlet left response electrode connection points Electrode connection points, n+1 entry electrode strips, and 2n+1 exit electrode strips, the n+1 entry electrode strips are connected to the entry excitation electrode and n through the entry excitation electrode connection point and the n entry response electrode connection points, respectively. The 2n+1 outlet electrode strips are connected to the outlet excitation motor and the n outlet right response electrodes through the outlet excitation electrode connection points, the n outlet right response electrode connection points and the n outlet left response electrode connection points, respectively. The electrodes are connected to the n outlet left response electrodes.

As a further improvement of the embodiment of the present invention, the electrical impedance detection layer is a conductive film, and laser cutting is performed on the conductive layer of the conductive film to obtain an entry excitation electrode, n entry response electrodes, exit excitation electrodes, and n exit right response electrodes And n exits left response electrodes, and make insulation between electrodes.

As a further improvement of the embodiment of the present invention, the detection unit further includes a detection chip connection layer, the detection chip connection layer is disposed above the multi-channel detection chips, and the detection chip connection layer is provided with an electromagnetic shielding area.

Compared with the prior art, the technical solution of the present invention has the following beneficial effects: the embodiment of the present invention provides a cancer cell sorting and detection device, which integrates the sorting and detection of cancer cells. In the embodiment of the present invention, the upper clamp and the lower clamp are used as the supporting frame of the whole device, and the sorting unit for sorting cancer cells from the cell fluid and the detection unit for electrical impedance detection of cancer cells are fixed to ensure that The sealing and connection reliability of the entire device. The sample liquid inlet of the upper fixture serves as the entrance of the entire device, and the detection liquid outlet of the lower fixture serves as the outlet of the entire device. The cell solution to be detected is passed into the sorting unit through the sample liquid inlet, and the sorting unit separates the cancer cells in the cell solution. Separated from leukocytes, the sorted cancer cells are input into the detection unit for detection, the detection result is obtained, and the detected detection solution is output from the detection solution outlet. The cancer cell sorting and detection device of this embodiment can separate and obtain cancer cells from a mixed solution of leukocytes and cancer cells, and at the same time quickly and accurately detect cancer cells. Sorting is carried out first, and then the detection device is used for detection, which is convenient and quick.

Description of drawings

1 is a schematic diagram of an assembly explosion of a cancer cell sorting and detection device according to an embodiment of the present invention;

2 is a schematic diagram of the assembly of a cancer cell sorting and detection device according to an embodiment of the present invention;

Fig. 3 is the structural representation of the upper clamp;

Figure 4 (1) is a schematic structural diagram of the first gas layer of the flow regulating valve, and Figure 4 (2) is a schematic structural diagram of the first liquid layer of the flow regulating valve;

Fig. 5 is the assembly explosion schematic diagram of adjustable concentrating chip;

Fig. 6(1) is a schematic structural diagram of the second gas layer of the adjustable concentrating chip, Fig. 6(2) is a structural schematic diagram of the second elastic layer of the adjustable concentrating chip, Fig. 6(3) is the first structural diagram of the adjustable concentrating chip Schematic diagram of the structure of the two liquid layers, Figure 6 (4) is a schematic diagram of the structure of the concentration layer of the adjustable concentration chip;

Fig. 7 is the structural representation of the left connecting piece;

Fig. 8 is the structural representation of the right connecting piece;

9 is a schematic structural diagram of a sorting chip;

10 is a schematic structural diagram of a detection chip connection layer;

11 is a schematic diagram of an assembly explosion of a multi-channel detection chip;

Figure 12 (1) is a schematic structural diagram of the electrical impedance detection layer of the multi-channel detection chip, Figure 12 (2) is a schematic structural diagram of the flow channel layer of the multi-channel detection chip, and Figure 12 (3) is the sealing layer of the multi-channel detection chip. Schematic diagram of the structure;

Figure 13 is a schematic structural diagram of a circuit connection board;

Fig. 14 is a schematic view of the structure of the lower clamp.

detailed description

The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings.

An embodiment of the present invention provides a cancer cell sorting and detection device. As shown in FIG. 1 and FIG. 2 , it includes an upper fixture 1 , a sorting unit, a detection unit, and a lower fixture 10 in order from top to bottom. The sorting unit is used for sorting cancer cells from the cell fluid, and the detection unit is used for electrical impedance detection of the cancer cells. The upper fixture 1 is provided with a sample liquid inlet 11 , and the lower fixture 10 is provided with a detection liquid outlet 103 . The sample liquid inlet 11 is communicated with the inlet of the sorting unit, the outlet of the sorting unit is communicated with the inlet of the detection unit, and the outlet of the detection unit is communicated with the detection liquid outlet 103 .

In the above-mentioned embodiment, the upper clamp 1 and the lower clamp 10 are used as the supporting frame of the whole device to fix the sorting unit and the detection unit, so as to ensure the sealing and connection reliability of the whole device. The sample liquid inlet 11 of the upper fixture 1 serves as the inlet of the entire device, and the detection liquid outlet 103 of the lower fixture 10 serves as the outlet of the entire device. The cell solution to be detected is input into the sorting unit from the sample liquid inlet 11, the sorting unit separates the cancer cells from the white blood cells in the cell solution, and the sorted cancer cells are input into the detection unit for detection, and the detection result is obtained. The liquid is output from the detection liquid outlet 103 .

The cancer cell sorting and detection device of the above embodiment can separate and obtain cancer cells from a mixed solution of leukocytes and cancer cells, and at the same time quickly and accurately detect cancer cells, and integrate the sorting and detection of cancer cells without the need for Sorting is carried out first, and then the detection device is used for detection, which is convenient and quick.

As a preferred example, the sorting unit includes a sorting chip 6. As shown in FIG. 9, the sorting chip 6 includes a chip body, and the chip body is provided with a spiral sorting flow channel 62, a sorting liquid flow channel 65 and a waste liquid flow channel 63. Both the separation liquid flow channel 65 and the waste liquid flow channel 63 are connected to the outlet of the spiral separation flow channel 62. The inlet 61 of the spiral-type sorting flow channel 62 communicates with the sample liquid inlet 11 , and the outlet 67 of the sorting liquid flow channel 65 communicates with the inlet of the detection unit.

The cell solution to be tested enters the spiral sorting flow channel 62 from the sample liquid inlet 11 through the inlet 61 of the spiral sorting flow channel 62 , and the cancer cells and white blood cells in the cell solution to be detected are subjected to the spiral sorting flow channel 62 . Inertial force and Dean force of different magnitudes, cancer cells and leukocytes are separated at the exit of the spiral-type sorting channel 62, the cancer cell solution enters the sorting liquid channel 65, and the leukocyte solution enters the waste liquid channel 63, and then enters the separation channel 63. The cancer cell solution in the liquid selection channel 65 enters the detection unit through the outlet 67 and the inlet of the detection unit. The above embodiment utilizes the helical flow channel to separate cancer cells and leukocytes, the structure is simple, the sorting process does not require human intervention, and the sorting efficiency is high.

As a preferred example, as shown in FIG. 1 and FIG. 2 , the sorting unit further includes a sorting liquid concentration adjusting member, the sorting liquid concentration adjusting member is arranged between the upper fixture 1 and the sorting chip 6 , and the sorting liquid concentration adjusting member is The liquid inlet is communicated with the outlet 67 of the sorting liquid flow channel 65, and the liquid outlet is communicated with the inlet of the detection unit.

In the above-mentioned embodiment, a sorting liquid concentration adjustment member is set to concentrate the concentration of cancer cells sorted by the sorting chip 6, and then the concentrated cancer cell solution is input into the detection device for detection, thereby improving the detection unit's performance. detection accuracy.

As a preferred example, the separation liquid concentration regulator includes an adjustable concentration chip 3. As shown in FIG. 5, the adjustable concentration chip 3 includes a second gas layer 31, a second elastic layer 32, a second gas layer 31, a second elastic layer 32, and a second gas layer 31, a second elastic layer 32, Two liquid layer 33 and concentrated layer 34. As shown in FIG. 6( 1 ), the second gas layer 31 is provided with a second gas flow channel 313 and a first conditioning waste liquid through hole 311 . As shown in FIG. 6( 2 ), the second elastic layer 32 is provided with a second adjusting waste liquid through hole 321 . As shown in FIG. 6( 3 ), the second liquid layer 33 is provided with a second liquid channel 332 . As shown in FIG. 5 , the second gas flow channels 313 and the second liquid flow channels 332 are arranged in a cross shape. As shown in FIG. 6(4), the concentrated layer 34 is provided with a spiral-type concentrated flow channel 342, a concentrated waste liquid flow channel 344 and a concentrated liquid flow channel 345. The outlet of the concentrate flow channel 342 is connected. The inlet 341 of the spiral-type concentrated flow channel 342 is communicated with the outlet 67 of the separation liquid flow channel 65, and the outlet 346 of the concentrated solution flow channel 345 is communicated with the inlet of the detection unit.

As shown in FIG. 3 , the upper fixture 1 is provided with a second regulating gas inlet 17 , a second regulating gas outlet 16 and a second regulating liquid outlet 15 , and the second regulating gas inlet 17 is communicated with the inlet 314 of the second gas flow channel 313 , The second conditioning gas outlet 16 communicates with the outlet 312 of the second gas flow channel 313 . As shown in FIG. 5 , the second adjustment liquid outlet 15 is communicated with the outlet 331 of the second liquid flow channel 332 through the first adjustment waste liquid through hole 311 and the second adjustment waste liquid through hole 321 , and the inlet of the second liquid flow channel 332 333 communicates with the outlet 343 of the concentrated waste liquid flow channel 344 . Preferably, the sorting liquid concentration adjusting member further includes a right connecting piece 5, as shown in FIG. The outlet 346 of the concentrated solution flow channel 345 is connected to the inlet of the detection unit through the pus through hole 52 through the separation liquid through hole 51 .

In the above embodiment, the cancer cell solution obtained by sorting by the sorting chip 6 enters the spiral concentrating flow channel 342 of the concentrating layer 34, and the sorting action of the spiral concentrating flow channel 342 further separates cancer cells and white blood cells to obtain: Cancer cell solution with higher concentration. The adjustable concentrating chip 3 is formed by stacking a control layer 31 , a second elastic layer 32 , a flow resistance layer 33 and a concentrating layer 34 from top to bottom. The control layer 31 is provided with a second gas flow channel 313, and the flow resistance layer 33 is provided with a second liquid flow channel 332. The second gas flow channel 313 and the second liquid flow channel 332 are arranged in a cross shape, and there is a space between them. layer second elastic layer 32 . The second liquid flow channel 332 is connected to the waste liquid flow channel 344 of the concentration layer 34 , so when the air pressure of the second gas flow channel 313 is increased, the gas in the second gas flow channel 313 will cause the second elastic layer 32 to deform , so that the cross-sectional area of the second liquid flow channel 332 is reduced, thereby changing the flow resistance of the second liquid flow channel 332 . The second liquid flow channel 332 is connected to the waste liquid flow channel 344 , which can change the flow resistance of the waste liquid flow channel 344 of the concentrated layer 34 and the concentrated liquid flow channel 345 of the concentrated chip, and can adjust the concentration of the cancer cell solution obtained by the concentrated liquid flow channel 345 . For cell solutions to be detected containing cancer cells and leukocytes of different concentrations, the adjustable concentration chip 3 can be used to sort and concentrate cancer cell solutions with the same concentration. For most electrical impedance detection chips, the concentration of the detected cell solution is often within a specific range, and this embodiment realizes the electrical impedance detection of cancer cells with a wider concentration of cell solution.

Considering that the adjustable concentration chip 3 is connected to the outlet 67 of the sorting liquid flow channel 65 of the sorting chip 6, when the flow resistance of the adjustable concentration chip 3 is changed by the air pressure control, the waste liquid flow channel of the sorting chip 6 is changed. The flow resistance of 63 and the separation liquid flow channel 65 will also change slightly, which in turn affects the separation efficiency of the separation chip 6 . In order to adjust the flow resistance change, preferably, between the upper fixture 1 and the sorting chip 6, a waste liquid flow regulating member is also provided, and the waste liquid flow regulating member is connected to the outlet 64 of the waste liquid flow channel 63 for adjusting The flow resistance of the waste liquid flow channel 63 ensures the sorting efficiency of the sorting chip 6 and the concentration efficiency of the adjustable concentration chip 3 .

As a preferred example, the waste liquid flow regulating member includes a flow regulating valve 2. As shown in FIG. 4 , the flow regulating valve 2 includes a first gas layer, a first elastic layer and a first liquid layer that are stacked in sequence from top to bottom. As shown in FIG. 4( 1 ), the first gas layer is provided with a first gas flow channel 22 and a third adjustment waste liquid through hole 27 . The first elastic layer is provided with a fourth adjusting waste liquid through hole. As shown in FIG. 4( 2 ), the first liquid layer is provided with a first liquid flow channel 25 . The first gas flow channel 22 and the first liquid flow channel 25 are arranged in a cross shape.

As shown in FIG. 3 , the upper fixture 1 is provided with a first regulating gas inlet 12 , a first regulating gas outlet 13 and a first regulating liquid outlet 14 , and the first regulating gas inlet 12 communicates with the inlet 21 of the first gas flow channel 22 , The first regulated gas outlet 13 communicates with the outlet 23 of the first gas flow channel 22 . The first adjustment liquid outlet 14 is communicated with the outlet 24 of the first liquid flow channel 25 through the third adjustment waste liquid through hole 27 and the fourth adjustment waste liquid through hole, and the inlet 26 of the first liquid flow channel 25 is connected to the separation chip 6 The outlet 64 of the waste liquid flow path 63 communicates with each other. Preferably, the waste liquid flow regulating member further includes a left connecting piece 4. As shown in FIG. 7, the left connecting piece 4 is provided with a waste liquid through hole 41, and the outlet 64 of the waste liquid flow channel 63 communicates with the first liquid through the waste liquid through hole 41. The inlet 26 of the liquid flow channel 25 communicates with each other.

The flow regulating valve 2 has a first gas layer, a first elastic layer and a first liquid layer that are stacked in sequence from top to bottom. The first gas layer is provided with a first gas flow channel 22, the first liquid layer is provided with a first liquid flow channel 25, and the first gas flow channel 22 and the first liquid flow channel 25 are arranged in a cross shape. The first liquid flow channel 25 is connected to the waste liquid flow channel 63 of the sorting chip 6 through the left connecting layer 4, so when the air pressure of the first gas flow channel is increased, the gas in the first gas flow channel will promote the first elasticity The layer is deformed, so that the cross-sectional area of the first liquid flow channel is reduced, thereby changing the flow resistance of the first liquid flow channel. However, since the first liquid flow channel is connected to the waste liquid flow channel 63 of the sorting chip 6 , the flow resistance of the first liquid flow channel changes will cause the flow resistance of the waste liquid flow channel 63 to change. The flow resistance of the waste liquid flow channel 63 is adjusted by the flow regulating valve 2 to ensure the sorting efficiency of the sorting chip 6 .

As a preferred example, the detection unit includes a multi-channel detection chip 8 and a circuit connection board 9 that are superimposed and arranged, and the multi-channel detection chip 8 is connected to the circuit connection board 9 . As shown in FIG. 11 , the multi-channel detection chip 8 includes an electrical impedance detection layer 81 and a flow channel layer 82 that are superimposed and arranged. The electrical impedance detection layer 81 is connected to the circuit connection board 9 , and the flow channel layer 82 is located between the electrical impedance detection layer 81 and the circuit. between the connecting plates 9.

As shown in FIG. 12 (2), the flow channel layer 82 is provided with an even distribution channel 822, n parallel detection channels 825 and a collection channel 829, and the inlet 821 of the equal distribution channel 822 is connected to the outlet of the sorting unit, The outlets of the equally divided flow channels 822 are respectively connected with the inlets of the n detection flow channels 825 , the outlets of the n detection flow channels 825 are connected with the inlets of the collection flow channels 829 , and the outlet 8210 of the collection flow channels 829 is connected with the detection liquid outlet of the lower fixture 10 . 103 connections. Among them, n represents an integer greater than or equal to 2. Preferably, n is 8.

As shown in FIG. 12(1), the electrical impedance detection layer 81 is provided with an inlet excitation electrode 813, n inlet response electrodes 812, outlet excitation electrodes 815, n outlet right response electrodes 814, n outlet left response electrodes 816, and a nth outlet left response electrode 816. A detection liquid through hole 811 . The inlet excitation electrode 813 corresponds to the inlet of the detection channel 825 , and the outlet excitation electrode 815 corresponds to the outlet of the detection channel 825 . The n inlet response electrodes are arranged at intervals along the extending direction of the inlet excitation electrode 813 and correspond to the n detection flow channels 825 one-to-one respectively. The n outlet right response electrodes 814 and the n outlet left response electrodes 816 are symmetrically arranged on both sides of the outlet excitation electrode 815 , and are arranged at intervals along the extension direction of the outlet excitation electrode 815 , respectively corresponding to the n detection channels 825 one-to-one. The first detection liquid through hole 811 equally divides the inlet 821 of the flow channel 822 to communicate with each other.

As shown in FIGS. 11-12 , the electrodes of the electrical impedance detection layer 81 correspond to the meanings of the detection flow channels of the flow channel layer 82 . The inlet excitation electrode 813 and the inlet response electrode 812 generate an electric field in each corresponding flow channel, and the electric field changes when a cell flows through the detection flow channel. The electric field changes caused by different cells are different, which can distinguish different types of cells. The parallel multi-channel detection flow channels and electrodes can improve the detection rate. The method for detecting cells by the exit excitation electrode 815, the exit right response electrode 814, and the exit left response electrode 816 is the same as this. Setting both the excitation electrode and the response electrode at the outlet and the inlet can improve the detection accuracy of cells.

Preferably, the multi-channel detection chip 8 further includes a sealing layer 83. As shown in FIG. 12(3), the sealing layer 83 is provided with a second detection liquid through hole 836, and the outlet 8210 of the collecting channel 829 passes through the second detection liquid through hole 836 communicates with the detection liquid outlet 103 of the lower jig 10 .

The sealing layer 83 mainly seals the detection flow channel of the flow channel layer 82 , and it seals the detection flow channel on the upper and lower sides together with the electrical impedance detection layer 81 . In addition, each detection electrode through hole provided in the sealing layer 83 corresponds to each excitation electrode and corresponding electrode of the electrical impedance detection layer 81, which is convenient for each electrode connection point of the circuit connection board 9 and each excitation electrode of the electrical impedance detection layer 81. electrodes are connected.

As a preferred example, as shown in FIG. 13 , the circuit connection board 9 is provided with inlet excitation electrode connection points 92 , n inlet response electrode connection points 91 , outlet excitation electrode connection points 96 , and n outlet right response electrode connection points 95 , n outlet left response electrode connection point 97, n+1 inlet electrode strips and 2n+1 outlet electrode strips, the n+1 inlet electrode strips are connected to each other through the inlet excitation electrode connection point 92 and the n inlet response electrode connection points 91, respectively. The inlet excitation electrode 813 is connected to the n inlet response electrodes 812, and the 2n+1 outlet electrode strips are connected to the outlet through the outlet excitation electrode connection point 96, the n outlet right response electrode connection point 95, and the n outlet left response electrode connection point 97, respectively. The excitation electrode 815, the n outlet right response electrodes 814, and the n outlet left response electrodes 816 are connected. The circuit connection board 9 also has a fourth detection liquid through hole 98 , and the outlet 8210 of the collecting channel 829 communicates with the detection liquid outlet 103 of the lower fixture 10 through the second detection liquid through hole 836 and the third detection liquid through hole 98 .

The flow channel layer 82 has 3n+2 first electrode through holes correspondingly, the sealing layer 83 has 3n+2 second electrode through holes correspondingly, and the 3n+2 electrodes pass through the 3n+2 first electrode through holes and 3n+2 second electrode through holes are connected to 3n+2 electrode strips through 3n+2 electrode connection points.

The circuit connection board 9 mainly transmits the electrical signals obtained by the multi-channel detection chip 8 to the external equipment, and the circuit connection points on it correspond to the excitation electrodes and response electrodes of the electrical impedance detection layer 81 respectively for signal transmission. In addition, an electromagnetic shielding area is also integrated inside the circuit connection board 9 , the size of this area is equal to the electromagnetic shielding area of the detection chip connection layer 7 , and the positions are coincident in the perspective of the top view. This area can ensure that the signal will not be interfered by external electromagnetic waves during the detection process, and improve the detection accuracy.

As a preferred example, the electrical impedance detection layer 81 is a conductive film, and laser cutting is performed on the conductive layer of the conductive film to obtain the inlet excitation electrodes 813, n inlet response electrodes 812, outlet excitation motors 815, n outlet right response electrodes 814 and n One outlet left the responsive electrode 816 and provided insulation between the electrodes.

The electrical impedance detection layer 81 may be a conductive film, and in this embodiment, the electrodes may be obtained by means of laser cutting or photolithography. Compared with the photolithography process, laser cutting is more convenient to obtain electrodes, with low processing cost and less time-consuming. The electrodes of the device are fabricated by a photolithography process, and the electrodes have higher precision.

As a preferred example, the detection unit further includes a detection chip connection layer 7 , as shown in FIG. 10 , the detection chip connection layer 7 is arranged above the multi-channel detection chip 8 , and the detection chip connection layer 7 is provided with an electromagnetic shielding area 71 and a fourth detection Liquid through hole 72 . The electromagnetic shielding area 71 can cover the detection area of the lower multi-channel detection chip 8 to form electromagnetic shielding to avoid mutual interference of electrical signals. The inlet 821 of the equally divided flow channel 822 communicates with the outlet of the sorting unit through the first detection liquid through hole 811 and the fourth detection liquid through hole 72 .

Preferably, as shown in FIG. 14, the lower fixture 10 is provided with a detection liquid inlet 101 and a detection liquid flow channel 102, the detection liquid inlet 101 is connected with the inlet of the detection liquid flow channel 102, and the outlet of the detection liquid flow channel 102 is connected with the detection liquid The outlet 103 is in communication, and the detection liquid inlet 101 is in communication with the outlet of the detection unit.

When using the cancer cell sorting and detection device of the above embodiment, the upper fixture 1 is respectively connected with the syringe containing the sample liquid, the waste liquid collection tube and the gas control device, which are used for sample injection and waste liquid collection of the device. The cell solution to be detected containing cancer cells and leukocytes enters the sorting chip 6 through the sample liquid inlet 11 of the upper fixture 1 , and the cancer cells and leukocytes are separated by the sorting chip 6 . The sorted cancer cells are guided to the adjustable concentration chip 3 through the sorting liquid flow channel 65 and the right connecting layer 5, and the cancer cells are concentrated through the adjustable concentration chip 3 and then guided through the right connecting layer 5 and the sorting chip 6 at most. The channel detection chip 8 realizes multi-channel electrical impedance signal detection of cancer cells, and the cancer cells detected by the electrical impedance are discharged through the fixture 10 under the device. Cancer cells are divided into eight channels in the multi-channel detection chip 8 to realize high-throughput electrical impedance detection of cancer cells, wherein the multi-channel detection chip 8 is realized by stacking multiple layers of thin film layers. An electromagnetic shielding area 71 is provided on the detection chip connection layer 7 , and the area of the area can cover the detection area of the lower multi-channel detection chip 8 to form electromagnetic shielding. The circuit connection board 9 is provided with a connection point corresponding to the detection electrode of each detection channel, so as to realize the transmission of electrical signals to the relevant hardware. In addition, an electromagnetic shielding area is also set inside the circuit connection board to avoid mutual interference of signals and realize multi-channel. fast and accurate detection. The leukocytes separated by the sorting chip 6 are guided to the flow regulating valve 2 through the waste liquid flow channel 63 and the left connecting layer 4 , and finally discharged through the upper fixture 1 . For the concentration layer 34 of the adjustable concentration chip 3, when the flow resistance of the waste liquid flow channel 344 of the concentration layer 34 and the flow resistance of the concentrated liquid flow channel 345 of the concentration chip are changed, the concentration ratio of the concentration layer 34 will be changed. That is, for solutions with different cell concentrations, changing the flow resistance of the waste liquid flow channel 344 and the concentrated liquid flow channel 345 of the concentrated chip can adjust the concentration of the cell solution obtained by the concentrated liquid flow channel 345. The adjustable concentration chip 3 is controlled by gas. change its flow resistance. The adjustable concentrating chip 3 is connected in series with the sorting liquid outlet 67 of the sorting chip 6. When the flow resistance of the adjustable concentrating chip 3 is changed by the air pressure control, the waste liquid flow channel 63 and the sorting liquid of the sorting chip 6 The flow resistance of the liquid selection channel 65 will also change slightly, thereby affecting the sorting efficiency of the sorting chip 6 . In order to adjust the flow resistance change, the flow regulating valve 2 is connected in series at the tail of the waste liquid flow channel 63 to ensure the sorting efficiency of the sorting chip 6 and the concentration efficiency of the adjustable concentration chip 3 . For sample liquids containing cancer cells and leukocytes of different concentrations, the flow resistance of the flow regulating valve 2 and the adjustable concentration chip 3 can be adjusted by air pressure control, and the cancer cell solution of the same concentration can be obtained by sorting and concentrating. For most electrical impedance detection chips, the concentration of the detected cell solution is often in a specific range. This embodiment realizes the electrical impedance detection of cancer cells with a wider concentration of cell solution, and the parallelism of the multi-channel detection channels is also realized. High-throughput and rapid cancer cell detection, in addition, the multi-channel detection chip 8 is fabricated by stacking multiple thin film layers, which facilitates the rapid fabrication of the electrical impedance detection chip and the integration with other microfluidic devices.

In the above embodiment, the upper fixture 1 and the lower fixture 10 are acrylic plates, the sample liquid inlet 11, the first regulating gas inlet 12, the first regulating gas outlet 13, the first regulating liquid outlet 14, the concentrated chip waste liquid outlet 15, the first regulating gas outlet 13, the The second regulated gas inlet 17 , the second regulated gas outlet 16 and the second regulated liquid outlet 15 are made of stainless steel.

In the above embodiment, the flow regulating valve 2 is made of silica gel and PVC plastic, and is fabricated by laser cutting and ion bonding technology. The control layer 31 and the concentration layer 34 of the adjustable concentration chip 3 are both in two-piece structure, the upper layer of the control layer 31 and the lower layer of the concentration layer 34 are PVC plastic, the lower layer of the control layer 31, the upper layer of the concentration layer 34, the second elastic layer 32. The flow resistance layer 33 is made of silica gel material. During production, the desired structure is engraved on the selected PVC substrate and silica gel substrate respectively with a laser, and then the encapsulation is completed by ion bonding technology. The material of the left connecting piece 4 and the right connecting piece 5 is double-sided tape. During production, the desired structure is engraved with a laser on the selected double-sided adhesive substrate. The sorting chip 6 has a three-piece structure, the middle layer is made of silica gel, and the outer two layers are made of PVC plastic. During production, the desired structure is engraved on the selected PVC substrate and silica gel substrate respectively with a laser, and then the encapsulation is completed by ion bonding technology.

In the above embodiment, the main body of the detection chip connection layer 7 is made of double-sided tape, and the electromagnetic shielding area 71 is inserted with aluminum foil to ensure electromagnetic shielding.

In the above-mentioned embodiment, the manufacturing material of the electrical impedance detection layer 81 of the multi-channel detection chip 8 is an ITO conductive film, a gold-plated PET film or other types of conductive films. The electrode shape is cut out of the layer and the electrodes are insulated, but the base material of the ITO film is not cut; the photolithography process is used to make the electrode of the gold-plated conductive film. The material of the flow channel layer 82 is AB double-sided tape, and its material characteristic is that the PET film is coated with adhesive colloid on both sides to ensure the connection of the two sides, and the required structure is obtained by laser cutting during production. The material of the sealing layer 83 is PVC, and laser cutting is used to obtain the required structure during manufacture.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above-mentioned specific embodiments, and the descriptions in the above-mentioned specific embodiments and the specification are only to further illustrate the principle of the present invention, without departing from the spirit and scope of the present invention, the present invention Various changes and modifications of the invention are also possible, all of which fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the claims and their equivalents.

Claims

An apparatus for sorting and detecting cancer cells, characterized in that it comprises an upper clamp (1), a sorting unit, a detection unit and a lower clamp (10) in sequence from top to bottom, and the sorting unit is used for removing cells from a cell fluid The cancer cells are sorted out, and the detection unit is used for electrical impedance detection of the cancer cells; the upper fixture (1) is provided with a sample liquid inlet (11), and the lower fixture (10) is provided with a detection liquid outlet (103) The sample liquid inlet (11) is communicated with the inlet of the sorting unit, the outlet of the sorting unit is communicated with the inlet of the detection unit, and the outlet of the detection unit is communicated with the detection liquid outlet (103).
The cancer cell sorting and detection device according to claim 1, wherein the sorting unit comprises a sorting chip (6), the sorting chip (6) comprises a chip body, and the chip body is provided with The spiral-type separation flow channel (62), the separation liquid flow channel (65) and the waste liquid flow channel (63), the separation liquid flow channel (65) and the waste liquid flow channel (63) are both connected with the spiral separation flow channel (63). The outlet of the sorting flow channel (62) is connected; the inlet (61) of the spiral sorting flow channel (62) is communicated with the sample liquid inlet (11), and the outlet (67) of the sorting liquid flow channel (65) is connected to the detection The inlet of the unit is connected.
The cancer cell sorting and detection device according to claim 2, characterized in that, the sorting unit further comprises a sorting liquid concentration regulator for concentrating the concentration of the cancer cells sorted by the sorting chip (6). ; The sorting liquid concentration regulator is arranged between the upper clamp (1) and the sorting chip (6), and the sorting liquid concentration regulator is respectively connected to the outlet (67 of the sorting liquid flow channel (65)) ) communicates with the inlet of the detection unit.
The cancer cell sorting and detection device according to claim 3, characterized in that, the sorting solution concentration regulator comprises an adjustable concentration chip (3), and the adjustable concentration chip (3) comprises a top-down a second gas layer (31), a second elastic layer (32), a second liquid layer (33) and a concentration layer (34) arranged in sequence;

The second gas layer (31) is provided with a second gas flow channel (313), the second liquid layer (33) is provided with a second liquid flow channel (332), and the second gas flow channel (313) and the second liquid flow channel (332) is arranged in a cross shape;

The concentrated layer (34) is provided with a spiral type concentrated flow channel (342), a concentrated waste liquid flow channel (344) and a concentrated liquid flow channel (345), and the concentrated waste liquid flow channel (344) and the concentrated liquid flow channel (345) are all connected with the outlet of the spiral concentrated flow channel (342); the inlet (341) of the spiral concentrated flow channel (342) is communicated with the outlet (67) of the separation liquid flow channel (65); the The outlet (346) of the concentrate flow channel (345) is communicated with the inlet of the detection unit;

The upper clamp (1) is provided with a second regulating gas inlet (17), a second regulating gas outlet (16) and a second regulating liquid outlet (15), and the second regulating gas inlet (17) is connected to the second gas The inlet (314) of the flow channel (313) communicates with the second regulating gas outlet (16) and the outlet (312) of the second gas channel (313), and the second regulating liquid outlet (15) communicates with the second regulating liquid outlet (15). The outlet (333) of the second liquid flow channel (332) is communicated, and the inlet (331) of the second liquid flow channel (332) is communicated with the outlet (343) of the concentrated waste liquid flow channel (344).
The cancer cell sorting and detection device according to claim 3, wherein a waste liquid flow regulating member is further provided between the upper clamp (1) and the sorting chip (6), and the waste liquid flow rate is adjusted The fitting is connected with the outlet (64) of the waste liquid flow channel (63), and is used to adjust the flow resistance of the waste liquid flow channel (63).
The cancer cell sorting and detection device according to claim 5, characterized in that, the waste liquid flow regulating member comprises a flow regulating valve (2), and the flow regulating valve (2) comprises a top-to-bottom stacking arrangement. a first gas layer, a first elastic layer and a first liquid layer, the first gas layer is provided with a first gas flow channel (22), and the first liquid layer is provided with a first liquid flow channel (25), The first gas flow channel (22) and the first liquid flow channel (25) are arranged in a cross shape;

The upper clamp (1) is provided with a first regulating gas inlet (12), a first regulating gas outlet (13) and a first regulating liquid outlet (14), and the first regulating gas inlet (12) is connected with the first gas The inlet (21) of the flow channel (22) is in communication, the first regulating gas outlet (13) is in communication with the outlet (23) of the first gas flow channel (22), and the first regulating liquid outlet (14) is in communication with the first regulating liquid outlet (14) The outlet (24) of a liquid flow channel (25) communicates with the inlet (26) of the first fluid flow channel (25) and the outlet (64) of the waste liquid flow channel (63) of the sorting chip (6) .
The cancer cell sorting and detection device according to claim 1, characterized in that the detection unit comprises a multi-channel detection chip (8) and a circuit connection board (9) that are superimposed and arranged, and the multi-channel detection chip (8) ) is connected to the circuit connection board (9); the multi-channel detection chip (8) comprises an electrical impedance detection layer (81) and a flow channel layer (82) arranged superimposed, and the electrical impedance detection layer (81) is connected to the circuit connection board (81). 9) connection, the flow channel layer (82) is located between the electrical impedance detection layer (81) and the circuit connection board (9);

The flow channel layer (82) is provided with an evenly divided flow channel (822), n parallelly arranged detection flow channels (825) and a collection flow channel (829). The outlets of the sorting units are connected, the outlets of the equally divided flow channels (822) are respectively connected with the inlets of the n detection flow channels (825), and the outlets of the n detection flow channels (825) are connected with the inlet of the collection flow channel (829) connected, the outlet of the collecting flow channel (829) is connected with the detection liquid outlet (103) of the lower fixture (10); n represents an integer greater than or equal to 2;

The electrical impedance detection layer (81) is provided with an inlet excitation electrode (813), n inlet response electrodes (812), an outlet excitation electrode (815), n outlet right response electrodes (814) and n outlet left response electrodes (816); the inlet excitation electrode (813) corresponds to the inlet of the detection channel (825), and the outlet excitation electrode (815) corresponds to the outlet of the detection channel (825); the n inlet response electrodes are excited along the inlet The extending directions of the electrodes (813) are arranged at intervals and correspond to the n detection flow channels (825) one-to-one respectively; the n outlet right response electrodes (814) and the n outlet left response electrodes (816) are symmetrically arranged at the outlet The two sides of the excitation electrode (815) are arranged at intervals along the extension direction of the outlet excitation electrode (815), respectively corresponding to the n detection flow channels (825) one-to-one.
The cancer cell sorting and detection device according to claim 7, wherein the circuit connection board (9) is provided with an inlet excitation electrode connection point (92), n inlet response electrode connection points (91), an outlet Excitation electrode connection points (96), n outlet right response electrode connection points (95), n outlet left response electrode connection points (97), n+1 inlet electrode strips (93) and 2n+1 outlet electrode strips (94), the n+1 inlet electrode strips (93) are connected to the inlet excitation electrode (813) and the n inlet response electrodes respectively through the inlet excitation electrode connection point (92) and the n inlet response electrode connection points (91) (812) connected, the 2n+1 outlet electrode strips (94) through outlet excitation electrode connection points (96), n outlet right response electrode connection points (95) and n outlet left response electrode connection points (97) It is respectively connected with the exit excitation motor (815), the n exit right response electrodes (814) and the n exit left response electrodes (816).
The cancer cell sorting and detection device according to claim 7, wherein the electrical impedance detection layer (81) is a conductive film, and laser cutting is performed on the conductive layer of the conductive film to obtain the entrance excitation electrode (813), n inlet response electrodes (812), outlet excitation electrodes (815), n outlet right response electrodes (814), and n outlet left response electrodes (816), with insulation between the electrodes.
The cancer cell sorting and detection device according to claim 7, wherein the detection unit further comprises a detection chip connection layer (7), and the detection chip connection layer (7) is arranged on the multi-channel detection chip (8). ), the detection chip connection layer (7) is provided with an electromagnetic shielding area (71).