A circulating tumor cell in vivo clearance system

Abstract

This invention provides an in vivo circulating tumor cell (CTC) clearance system, relating to the field of medical device technology. The in vivo CTC clearance system provided in this invention utilizes switching between three pathways: a CTC separation pathway, a CTC electroporation pathway, and a cell debris removal pathway. Based on a metal filter membrane, it continuously separates CTCs in vivo and periodically performs electroporation to break down and remove the collected CTCs. This avoids clogging of the filter pores due to continuous metal filter operation, allowing for continuous, periodic operation to clear all existing and newly generated circulating tumor cells (CTCs) in the body.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and in particular to a circulating tumor cell in vivo clearance system. Background Technology

[0002] Tumor cells shed from the primary tumor into the peripheral blood are called circulating tumor cells (CTCs). These cells survive in the peripheral blood, extravasate, and colonize, forming new metastatic lesions, which is one of the key factors mediating tumor metastasis. Clearing circulating tumor cells (CTCs) from the peripheral blood holds promise for reducing the risk of tumor metastasis and could be used as an adjunct to chemotherapy and radiotherapy. However, this approach faces two challenges: 1. How to isolate circulating tumor cells from the entire bloodstream; 2. How to clear circulating tumor cells. Current research has stopped at sampling-based isolation of CTCs or clearing small amounts of CTCs in vivo, without achieving treatment targeting peripheral blood CTCs, mainly due to the difficulty in continuously processing the patient's entire blood system.

[0003] One approach provided in related technologies is to use dielectrophoresis to separate CTCs and then electroporation to remove the cells. However, this is a microfluidic circulating tumor cell in vivo removal system, which is greatly limited in terms of processing speed and volume. It is only suitable for blood collection scenarios and cannot efficiently remove all CTCs in the in vivo circulating tumor cell in vivo removal system, thus failing to minimize the risk of tumor metastasis.

[0004] Another approach provided in the related technologies is a method based on bioaffinity and in vivo capture and removal of CTCs. However, either the capture system has limited capacity and needs to be frequently removed and replaced, or it needs to ensure that the cells and the device have sufficient reaction time in the blood flow. Therefore, the processing throughput is limited and neither can meet the function of continuously removing all CTCs. Summary of the Invention

[0005] This invention provides an in vivo system for clearing circulating tumor cells, enabling continuous and efficient removal of circulating tumor cells from the body.

[0006] This invention provides an in vivo system for clearing circulating tumor cells, the system comprising the following components: a first peristaltic pump, a first solenoid valve, a clamp loaded with a metal filter membrane, a second solenoid valve, a second peristaltic pump, an electroporation buffer reservoir, a waste liquid reservoir, and a high-voltage pulse power supply;

[0007] The components are connected by silicone tubing to allow blood or electroporation buffer to flow. The first and second solenoid valves are used to control the switching of the circulating tumor cell separation pathway, the circulating tumor cell electroporation pathway, and the cell debris clearance pathway.

[0008] The circulating tumor cell separation pathway includes a connected artery of the living subject, a first peristaltic pump, a first solenoid valve, a clamp loaded with a metal filter membrane, a second solenoid valve, and a vein of the living subject.

[0009] The circulating tumor cell electroporation pathway includes a connected electroporation buffer reservoir, a second peristaltic pump, a first solenoid valve, a clamp loaded with a metal filter membrane, a second solenoid valve, and a waste liquid reservoir; the clamp loaded with the metal filter membrane is provided with a first platinum mesh electrode and a second platinum mesh electrode, and the first platinum mesh electrode and the second platinum mesh electrode are connected to a high-voltage pulse power supply through wires;

[0010] The cell debris removal pathway includes a connected living subject artery, a first peristaltic pump, a first solenoid valve, a clamp loaded with a metal filter membrane, a second solenoid valve, and a waste liquid storage tube.

[0011] Optionally, the metal filter membrane is a nickel porous membrane containing more than 270,000 filter pores.

[0012] Optionally, the area of ​​the nickel porous membrane is 1 cm2, the pore diameter is 8-10 μm, the pore spacing is 4-5 μm, and the pore shape is regular hexagonal.

[0013] Optionally, the fixture loaded with the metal filter membrane includes: a first fixture body, a second fixture body, a first pad, a second pad, a first platinum mesh electrode, a second platinum mesh electrode, and a buckle. The first platinum mesh electrode and the second platinum mesh electrode are led out and connected to a high-voltage pulse power supply through wires. The first pad and the second pad are used to fix the first platinum mesh electrode and the second platinum mesh electrode, respectively, and to control the electrode spacing between the first platinum mesh electrode and the second platinum mesh electrode. The metal filter membrane is sandwiched between the first pad and the second pad.

[0014] Optionally, during the process of establishing the circulating tumor cell separation pathway, the first peristaltic pump draws blood from the living subject's artery, and the blood flows through the first solenoid valve, the clamp loaded with a metal filter membrane, and the second solenoid valve before being returned to the living subject's vein. The metal filter membrane captures circulating tumor cells from the flowing blood of the living subject.

[0015] Optionally, during the process of establishing the electroporation pathway for circulating tumor cells, the second peristaltic pump draws electroporation buffer from the electroporation buffer reservoir. The electroporation buffer then flushes the clamp after passing through the first solenoid valve, pushing out any remaining blood on the metal filter membrane. The blood then flows into the waste liquid reservoir through the second solenoid valve, and the electroporation buffer fills the clamp, forming a uniform electroporation buffer medium. The first platinum mesh electrode and the second platinum mesh electrode, connected to a high-voltage pulse power supply, form an electric field on both sides of the metal filter membrane, completing the cell electroporation.

[0016] Optionally, during the process of establishing the cell debris removal pathway, the first peristaltic pump draws blood from the living subject's artery, and the blood then flushes the clamp after passing through the first solenoid valve, discharging the waste liquid of the electroporation buffer into the waste liquid storage tube, the waste liquid containing cell debris broken during the cell electroporation process.

[0017] Optionally, the system further includes: a path switching control device, which is connected and communicates with the first peristaltic pump, the second peristaltic pump, the first solenoid valve, and the second solenoid valve respectively based on a serial communication protocol. The path switching control device includes:

[0018] The first switching module is used to control the first solenoid valve and the second solenoid valve to switch to the connected state of the circulating tumor cell separation pathway, and to control the operation of the first peristaltic pump.

[0019] The second switching module is used to control the first solenoid valve and the second solenoid valve to switch to the state of connected electroporation pathway for circulating tumor cells, and to control the operation of the second peristaltic pump.

[0020] The third switching module is used to control the first and second solenoid valves to switch to the cell debris removal pathway connection state and to control the first peristaltic pump to work.

[0021] Optionally, the throughput of blood or electroporation buffer in the system is 1 ml / min.

[0022] Optionally, the nickel porous membrane is prepared according to the following steps:

[0023] Sputter an aluminum adhesion layer onto the silicon wafer surface;

[0024] A copper seed layer is sputtered onto the surface of the adhesion layer;

[0025] A micropillar array was formed on the surface of the copper seed layer by photolithography and development using AZ4620 photoresist.

[0026] Nickel is electroplated to grow nickel on the surface of the copper seed layer, forming a porous membrane structure;

[0027] The photoresist was washed away, and the copper was etched to obtain a nickel porous film.

[0028] The circulating tumor cell in vivo clearance system provided in this embodiment of the invention continuously separates circulating tumor cells in vivo based on a metal filter membrane by switching between three pathways: circulating tumor cell separation pathway, circulating tumor cell electroporation pathway, and cell debris clearance pathway. It also periodically electroporates and breaks down and removes the collected circulating tumor cells, avoiding continuous blockage of the filter pores by the metal filter membrane. Thus, it can work continuously and periodically to remove all existing and newly generated CTCs in vivo. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a schematic diagram of the structure of the circulating tumor cell in vivo clearance system provided in this embodiment of the invention;

[0031] Figure 2 This is a disassembly diagram of the fixture with a metal filter membrane loaded in the circulating tumor cell in vivo clearance system provided in this embodiment of the invention.

[0032] Figure 3 This is a schematic diagram of the manufacturing process of the metal filter membrane in the circulating tumor cell in vivo clearance system provided in this embodiment of the invention;

[0033] Figure 4 These are physical images, optical microscope images, and electron microscope images of the metal filter membrane in the circulating tumor cell in vivo clearance system provided in this embodiment of the invention.

[0034] Reference numerals: 1. Living object; 2. First peristaltic pump; 3. First solenoid valve; 4. Fixture with metal filter membrane; 5. First fixture body; 6. Second fixture body; 7. First platinum mesh electrode; 8. Second platinum mesh electrode; 9. First gasket; 10. Second gasket; 11. Metal filter membrane; 12. Buckle; 13. First wire; 14. Second wire; 15. Second solenoid valve; 16. Second peristaltic pump; 17. Electroporation buffer solution reservoir; 18. Waste liquid reservoir; 19. High-voltage pulse power supply. Detailed Implementation

[0035] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] In this embodiment of the invention, to remove all circulating tumor cells (CTCs) from peripheral blood, the following considerations are taken into account: firstly, whole-body blood processing is required to separate all CTCs; secondly, the separated CTCs also need to be rapidly removed. Therefore, this embodiment of the invention provides an in vivo circulating tumor cell removal system, specifically, as follows: Figure 1 As shown, it illustrates a schematic diagram of the structure of an in vivo circulating tumor cell clearance system according to an embodiment of the present invention.

[0037] In this embodiment of the invention, the circulating tumor cell in vivo clearance system includes the following components: a first peristaltic pump 2, a first solenoid valve 3, a clamp 4 loaded with a metal filter membrane, a second solenoid valve 15, a second peristaltic pump 16, an electroporation buffer reservoir 17, a waste liquid reservoir 18, and a high-voltage pulse power supply 19.

[0038] In this embodiment of the invention, the various components are connected via silicone tubing to allow the flow of blood or electroporation buffer from a living subject. During actual operation, the system operates in three connection states between the components: a circulating tumor cell separation pathway, a circulating tumor cell electroporation pathway, and a cell debris removal pathway. Specifically, the switching between these pathways can be controlled by the first and second solenoid valves.

[0039] Specifically, the circulating tumor cell separation pathway includes an artery connected to the living subject 1, a first peristaltic pump 2, a first solenoid valve 3, a clamp 4 loaded with a metal filter membrane, a second solenoid valve 15, and a vein of the living subject 1.

[0040] In this embodiment of the invention, during the process of connecting the circulating tumor cell separation pathway, the first peristaltic pump 2 draws blood from the living subject's artery, and the blood flows through the first solenoid valve 3, the clamp 4 loaded with a metal filter membrane, and the second solenoid valve 15 before being returned to the living subject's vein. The metal filter membrane captures circulating tumor cells from the flowing blood of the living subject.

[0041] Specifically, the circulating tumor cell electroporation pathway includes a connected electroporation buffer reservoir, a second peristaltic pump 16, a first solenoid valve 3, a clamp 4 loaded with a metal filter membrane, a second solenoid valve 15, and a waste liquid reservoir 18; the clamp 4 loaded with the metal filter membrane is provided with a first platinum mesh electrode and a second platinum mesh electrode, and the first platinum mesh electrode and the second platinum mesh electrode are connected to the waste liquid reservoir 19 by wires.

[0042] In this embodiment of the invention, during the process of establishing the electroporation pathway for circulating tumor cells, the second peristaltic pump 16 draws electroporation buffer from the electroporation buffer reservoir tube. The electroporation buffer then flushes the clamp after passing through the first solenoid valve 3, pushing out the remaining blood on the metal filter membrane. The blood then flows into the waste liquid reservoir tube 18 through the second solenoid valve 15, and the electroporation buffer fills the clamp, forming a uniform electroporation buffer medium. The first platinum mesh electrode and the second platinum mesh electrode, connected to the waste liquid reservoir tube 19, form an electric field on both sides of the metal filter membrane, completing the cell electroporation.

[0043] Specifically, the cell debris removal pathway includes a connected living subject artery, a first peristaltic pump 2, a first solenoid valve 3, a clamp 4 loaded with a metal filter membrane, a second solenoid valve 15, and a waste liquid storage tube 18.

[0044] In this embodiment of the invention, during the process of connecting the cell debris removal pathway, the first peristaltic pump 2 draws blood from the living subject's artery, and the living subject's blood flushes the clamp after passing through the first solenoid valve 3, and discharges the waste liquid of the electroporation buffer into the waste liquid storage pipe 18, the waste liquid containing cell debris broken during the cell electroporation process.

[0045] Specifically, in this embodiment of the invention, the living subject can be a person or animal with CTCs present in peripheral blood.

[0046] In this embodiment of the invention, each component of the system can be disinfected before the system is actually run. The specific disinfection method can be a commonly used clinical disinfection method, such as alcohol disinfection.

[0047] In practical applications, a cannulation procedure can be performed on a live subject (e.g., a rat) to draw out the arteries and veins. Specifically, cannulation can be performed using the carotid artery and jugular vein, or the femoral artery and jugular vein. The cannula is then connected to the system via a silicone tube. Specifically, cannulation can be performed using the femoral artery and jugular vein. The femoral artery is connected to the inlet of the circulating tumor cell separation pathway of the system, so that the blood of the live subject can be drawn out through the first peristaltic pump 2. The jugular vein is connected to the outlet of the circulating tumor cell separation pathway, so that the blood filtered to remove CTCs can be returned to the live animal through the second solenoid valve 15.

[0048] Specifically, during the actual operation of the system, after connecting the system to the live animal, the first solenoid valve 3 and the second solenoid valve 15 can be switched to the circulating tumor cell separation pathway, and the first peristaltic pump 2 can be turned on. During the operation of the circulating tumor cell separation pathway, CTCs in the peripheral blood of the live subject will be captured on the surface of the metal filter membrane, while smaller red blood cells, white blood cells, and other blood components will be returned to the body. After the circulating tumor cell separation pathway has been running for a period of time (the specific time can be adjusted according to the actual clinical situation, for example, 1 hour), the first peristaltic pump 2 can be turned off, and the first solenoid valve 3 and the second solenoid valve 15 can be switched to the circulating tumor cell electroporation pathway. The second peristaltic pump 16 can be turned on to draw out the electroporation buffer solution. After passing through the first solenoid valve 3, the buffer solution flushes the clamp, pushes out the remaining blood, and flows into the waste liquid storage tank through the second solenoid valve 15, while filling the entire clamp to form a uniform electroporation liquid medium. Then, the second peristaltic pump 16 is turned off, and the waste liquid storage tube 19 is turned on. An electric field is formed on both sides of the metal filter membrane through wires and platinum mesh electrodes (the electric field strength and pulse width can be adjusted according to the actual clinical situation; for example, the electric field strength can be greater than 200V / mm, and the pulse width can be 10ms), and multiple pulses are applied (for example, 10 times). Under the action of the electric field, the cell membrane will be compressed to the point of collapse. Then, the first peristaltic pump 2 is turned on, and the first solenoid valve 3 and the second solenoid valve 15 are switched to the cell debris removal pathway, and blood is drawn out again. The blood drains the waste liquid of the electroshock buffer into the waste liquid storage tank. Some of the damaged cell debris will be discharged with the liquid, and the blocked membrane pores will reopen. After the waste liquid is discharged, the first solenoid valve 3 and the second solenoid valve 15 are quickly switched to the circulating tumor cell separation pathway, and the blood flows back into the rat through the reinfusion pathway to start the next cycle.

[0049] Specifically, the time interval from shutting down the first peristaltic pump 2 to starting the next cycle can be controlled within 10 minutes.

[0050] In this embodiment of the invention, through periodic pathway switching, the system can achieve 24-hour uninterrupted CTC removal in live animals, so as to continuously remove all existing and newly generated CTCs in the body.

[0051] Specifically, after a course of treatment is completed, the instrument can be paused, the metal filter membrane can be removed, and a new metal filter membrane can be replaced.

[0052] In this embodiment of the invention, the first solenoid valve 3 can control the liquid in the silicone tubing to move from the direction of the first peristaltic pump 2 to the direction of the clamp or from the direction of the second peristaltic pump 16 to the direction of the clamp, and the second solenoid valve 15 can control the liquid in the silicone tubing to move from the direction of the clamp to the direction of the waste liquid storage tube or from the direction of the clamp to the direction of the living object, thereby controlling the switching of the circulating tumor cell separation pathway, the circulating tumor cell electroporation pathway and the cell debris clearance pathway through the first solenoid valve 3 and the second solenoid valve 15.

[0053] In this embodiment of the invention, a disassembled diagram of the fixture containing the metal filter membrane is shown below. Figure 2 The fixture loaded with the metal filter membrane includes: a first fixture body 5, a second fixture body 6, a first pad 9, a second pad 10, a first platinum mesh electrode 7, a second platinum mesh electrode 8, and a buckle 12. The first fixture body 5 and the second fixture body 6 are the upper and lower halves of the fixture body, respectively. The first platinum mesh electrode 7 is led out through a first wire 13, and the second platinum mesh electrode 8 is led out through a second wire 14, and are respectively connected to a high-voltage pulse power supply 19. The first pad 9 and the second pad 10 are used to fix the first platinum mesh electrode 7 and the second platinum mesh electrode 8, and control the electrode spacing between the first platinum mesh electrode 7 and the second platinum mesh electrode 8. A metal filter membrane 11 is sandwiched between the first pad 9 and the second pad 10, and the buckle 12 fixes the entire fixture.

[0054] In this embodiment of the invention, the metal filter membrane can be a nickel porous membrane containing more than 270,000 filter pores, and the area of ​​the nickel porous membrane is 1 cm². 2 The filter pore diameter is 8-10μm, the pore spacing is 4-5μm, and the pore shape is regular hexagonal.

[0055] The nickel porous membrane described in this embodiment of the invention is prepared according to the following steps:

[0056] Step 1: Sputter an aluminum adhesion layer onto the silicon wafer surface.

[0057] Step 2: Sputter a copper seed layer onto the surface of the adhesion layer.

[0058] Step 3: A micropillar array is formed on the surface of the copper seed layer using AZ4620 photoresist.

[0059] Step four: Electroplating nickel to grow nickel on the surface of the copper seed layer, forming a porous membrane structure.

[0060] Step 5: Wash away the photoresist, etch copper, and obtain a nickel porous film.

[0061] Figure 3The fabrication process of the nickel porous film used in this embodiment of the invention is illustrated. (a) shows the sputtering of an aluminum adhesion layer onto a silicon wafer surface; (b) shows the sputtering of a copper seed layer onto the adhesion layer surface; (c) shows the formation of a micropillar array after photolithography and development using AZ4620 photoresist; (d) shows nickel electroplating: nickel is grown on the surface of the copper seed layer to form a porous film structure; and (e) shows the removal of the photoresist and etching of copper to obtain the nickel porous film. The fabricated porous film has an area of ​​1 cm². 2 The pore diameter is 8-10 μm, and the pore spacing is 4-5 μm. This metal filter membrane has high mechanical strength and can operate continuously at a flux of 1 ml / min.

[0062] Figure 4 The images shown are physical images, optical microscope images, and electron microscope images of the metal filter membrane used in the above embodiments.

[0063] In this embodiment of the invention, a nickel-based high-porosity microporous filter membrane, fabricated using micro-electro-mechanical system (MEMS) technology, can be applied to the circulating tumor cell (CTC) in vivo clearance system provided in this embodiment. Specifically, a femoral artery-jugular vein cannulation procedure can be performed on a living subject, and the system's operating state can be switched to the CTC separation pathway. A blood flow pathway is formed using a first peristaltic pump and a silicone tubing, flowing through a clamp containing the metal filter membrane. After operating for a certain period with the CTC separation pathway connected, circulating tumor cells (CTCs) are trapped on the metal filter membrane (a very small number of larger white blood cells may also be trapped). Therefore, this embodiment of the invention also includes a feature that can periodically switch the system's operating state to the CTC electroporation pathway connection to electroporate and break the trapped cells (CTCs and a very small number of larger white blood cells) on the metal filter membrane. Specifically, in this embodiment of the invention, a platinum mesh electrode is inserted into the clamp containing the filter membrane, and high voltage is applied through lead-out wires and a high-voltage pulse power supply to break the cells on the membrane. Next, switch the system to the cell debris removal pathway connection and flush the broken cells into the waste liquid reservoir along with the buffer solution. After removing the broken cell debris, the metal filter membrane can be considered to have reset its working state and can continue the next cycle for CTC separation.

[0064] In this embodiment of the invention, the metal filter membrane prepared by the above process has high mechanical strength and can work continuously at a flux of 1 ml / min, thereby enabling it to work in conjunction with the circulating tumor cell in vivo clearance system to continuously clear CTCs in vivo.

[0065] In this embodiment of the invention, based on the coordination of three working states of the circulating tumor cell (CTC) in vivo clearance system and a high-mechanical-strength metal filter membrane, a membrane filtration method can be used to capture circulating tumor cells (CTCs). This method has very little limitation on the blood velocity within the system, allowing for relatively rapid processing of peripheral blood. In fact, in practical applications, as long as the blood flow velocity within the system is not fast enough to cause CTC deformation, allowing them to be deformed and squeezed through the filter pores, the CTC capture function can be achieved. Based on this, this embodiment of the invention includes a first peristaltic pump 2 to control the blood extraction speed from the living subject, thereby achieving a blood processing rate of 1 ml per minute.

[0066] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0067] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0068] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0069] The above provides a detailed description of the circulating tumor cell in vivo clearance system provided by the present invention. Specific examples have been used to illustrate the principle and implementation of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation and application scope based on the idea of ​​the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A circulating tumor cell in vivo clearance system, characterized in that, The system includes the following components: a first peristaltic pump, a first solenoid valve, a clamp loaded with a metal filter membrane, a second solenoid valve, a second peristaltic pump, an electroporation buffer reservoir, a waste liquid reservoir, and a high-voltage pulse power supply. The components are connected by silicone tubing to allow blood or electroporation buffer to flow. The first and second solenoid valves are used to control the switching of the circulating tumor cell separation pathway, the circulating tumor cell electroporation pathway, and the cell debris clearance pathway. The circulating tumor cell separation pathway includes a connected artery of the living subject, a first peristaltic pump, a first solenoid valve, a clamp loaded with a metal filter membrane, a second solenoid valve, and a vein of the living subject. The circulating tumor cell electroporation pathway includes a connected electroporation buffer reservoir, a second peristaltic pump, a first solenoid valve, a clamp loaded with a metal filter membrane, a second solenoid valve, and a waste liquid reservoir; the clamp loaded with the metal filter membrane is provided with a first platinum mesh electrode and a second platinum mesh electrode, and the first platinum mesh electrode and the second platinum mesh electrode are connected to a high-voltage pulse power supply through wires; The cell debris removal pathway includes a connected living subject artery, a first peristaltic pump, a first solenoid valve, a clamp loaded with a metal filter membrane, a second solenoid valve, and a waste liquid storage tube.

2. The circulating tumor cell in vivo clearance system according to claim 1, characterized in that, The metal filter membrane is a nickel porous membrane, which contains more than 270,000 filter pores.

3. The circulating tumor cell in vivo clearance system according to claim 2, characterized in that, The area of ​​the nickel porous membrane is 1 cm². 2 The filter pore diameter is 8-10μm, the pore spacing is 4-5μm, and the pore shape is regular hexagonal.

4. The circulating tumor cell in vivo clearance system according to claim 1, characterized in that, The fixture loaded with the metal filter membrane includes: a first fixture body, a second fixture body, a first pad, a second pad, a first platinum mesh electrode, a second platinum mesh electrode, and a buckle. The first platinum mesh electrode and the second platinum mesh electrode are led out and connected to a high-voltage pulse power supply through wires. The first pad and the second pad are used to fix the first platinum mesh electrode and the second platinum mesh electrode, respectively, and to control the electrode spacing between the first platinum mesh electrode and the second platinum mesh electrode. The metal filter membrane is sandwiched between the first pad and the second pad.

5. The circulating tumor cell in vivo clearance system according to claim 1, characterized in that, During the process of establishing the circulating tumor cell separation pathway, the first peristaltic pump draws blood from the living subject's artery. The blood then flows through the first solenoid valve, a clamp equipped with a metal filter membrane, and the second solenoid valve before being returned to the living subject's vein. The metal filter membrane captures circulating tumor cells from the flowing blood of the living subject.

6. The circulating tumor cell in vivo clearance system according to claim 1, characterized in that, During the process of establishing the electroporation pathway for circulating tumor cells, the second peristaltic pump draws electroporation buffer from the electroporation buffer reservoir. The electroporation buffer then flushes the clamp after passing through the first solenoid valve, pushing out any remaining blood on the metal filter membrane. The blood then flows into the waste liquid reservoir through the second solenoid valve, and the electroporation buffer fills the clamp, forming a uniform electroporation buffer medium. The first and second platinum mesh electrodes, connected to a high-voltage pulse power supply, create an electric field on both sides of the metal filter membrane, completing the cell electroporation.

7. The circulating tumor cell in vivo clearance system according to claim 1, characterized in that, During the process of establishing the cell debris removal pathway, the first peristaltic pump draws blood from the living subject's artery. The blood then passes through the first solenoid valve and flushes the clamp, discharging the waste liquid of the electroporation buffer into the waste liquid storage tube. The waste liquid contains cell debris broken during the cell electroporation process.

8. The circulating tumor cell in vivo clearance system according to claim 1, characterized in that, The system further includes: a path switching control device, which is connected and communicates with the first peristaltic pump, the second peristaltic pump, the first solenoid valve, and the second solenoid valve respectively based on a serial communication protocol. The path switching control device includes: The first switching module is used to control the first solenoid valve and the second solenoid valve to switch to the connected state of the circulating tumor cell separation pathway, and to control the operation of the first peristaltic pump. The second switching module is used to control the first solenoid valve and the second solenoid valve to switch to the state of connected electroporation pathway for circulating tumor cells, and to control the operation of the second peristaltic pump. The third switching module is used to control the first and second solenoid valves to switch to the cell debris removal pathway connection state and to control the first peristaltic pump to work.

9. The circulating tumor cell in vivo clearance system according to any one of claims 1-8, characterized in that, The throughput of blood or electroporation buffer in the system is 1 ml / min.

10. The circulating tumor cell in vivo clearance system according to claim 2, characterized in that, The nickel porous membrane was prepared according to the following steps: Sputter an aluminum adhesion layer onto the silicon wafer surface; A copper seed layer is sputtered onto the surface of the adhesion layer; A micropillar array was formed on the surface of the copper seed layer by photolithography and development using AZ4620 photoresist. Nickel is electroplated to grow nickel on the surface of the copper seed layer, forming a porous membrane structure; The photoresist was washed away, and the copper was etched to obtain a nickel porous film.

Images