A highly sensitive detection chip for tumor cells, preparation method and usage method

Abstract

The present invention provides a highly sensitive detection chip for tumor cells, comprising a control membrane, a chip membrane and an electrode plate; the chip membrane includes a plurality of culture tanks and a plurality of flow channels; the number of flow channels is the same as the number of culture tanks, and a limiting member for limiting a single tumor cell is provided in each culture tank; the control membrane includes a plurality of control structures for controlling the on-off of the flow channels, and each control structure includes a pair of air ports and an air flow channel. When gas enters the air ports, the air flow channel expands and squeezes the flow channel, causing the flow channel to form a closure; when the gas is discharged from the air ports, the air flow channel contracts, causing the flow channel to form a passage; a plurality of three-electrode systems for detecting the change of liquid current in the culture tank are provided on the electrode plate; the present invention realizes the detection of the ROS level of tumor cells on a single chip, and realizes highly sensitive ROS level analysis without damaging the cells, improving the efficiency of anti-tumor drug development and the research on the mechanism of drug action.

Description

Technical Field

[0001] The present invention relates to the technical field of bio-microfluidic chips, and in particular, to a highly sensitive detection chip for tumor cells, a preparation method and a usage method thereof. Background Art

[0002] Reactive Oxygen Species (hereinafter referred to as ROS) are a class of free radical active substances, mainly including superoxide anion (O2·-), hydrogen peroxide (H2O2), hydroxyl radical (·OH) and singlet oxygen (1O2).

[0003] Compared with normal cells, tumor cells are in a higher oxidation state and are more susceptible to exogenous oxidation. Disrupting the ROS balance in tumor cells can induce their apoptosis and achieve the purpose of eliminating tumor cells. Therefore, tumor treatment based on ROS is currently recognized as an effective tumor treatment method.

[0004] The concentration change of ROS is an important detection index for the development of anti-tumor drugs and the study of treatment mechanisms. Detecting the ROS level of single tumor cells can more deeply explore the effect of anti-tumor drugs on heterogeneous single tumor cells. Currently, the emerging single-cell ROS detection methods such as mass spectrometry analysis, fluorescence analysis technology, capillary electrophoresis separation method, etc. either cannot achieve in-situ detection of living cells, or have low sensitivity, or are single-channel and single-index detection, or rely on large-scale instruments resulting in high costs, and it is difficult to meet the requirements of in-situ, dynamic and highly sensitive detection of ROS in single living cells.

[0005] Among them, the electrochemical analysis method is very suitable for online real-time dynamic detection of single-cell free radicals due to its simple equipment requirements, low cost, high sensitivity, fast response, no need for labeling and easy integration. For example, patent document CN115201309A discloses a hydroxyl radical electrochemical sensor. A molecularly imprinted polymer was prepared on the surface of a g-C3N4 / MWCNTs-COOH modified electrode by bulk polymerization method, and differential pulse voltammetry was used, with a detection limit of up to 3.2×10-9mol / L. For example, patent document CN114527185A discloses a hydrogen peroxide sensor based on a few-layer graphene composite material loaded with copper and silver. Cyclic voltammetry was used for detection. Its starting potential is positive, the reduction current is large, and the sensitivity is higher at a smaller voltage, with excellent sensing performance. The above methods have achieved highly sensitive detection of reactive oxygen species, but have not achieved the detection of multiple free radicals in single cells, and the electrode integration is not high.

[0006] In addition to the above detection methods, microfluidic chips have shown obvious advantages in single-cell in-situ detection because the size of their functional units is comparable to that of cells and it is easy to integrate other detection methods. For example, in patent document CN114540182A, a microfluidic system for detecting circulating tumor cell secretions at the single-cell level was developed. Through a primary spiral structure and a secondary micro-droplet formation structure, the enrichment and detection of tumor single-cell secretions were realized. Compared with the detection of population cell secretions, it provides a new analysis system for cell heterogeneity analysis, but lacks quantitative analysis and does not meet the requirements for the in-situ dynamic high-sensitivity detection of ROS in single tumor cells. Summary of the Invention

[0007] The problem solved by the present invention is to provide a high-sensitivity detection chip for tumor cells, a preparation method and a usage method, which can integrate a microfluidic and electrode system, realize the detection of the ROS level of tumor cells on a single chip, and achieve high-sensitivity ROS level analysis without destroying cells, greatly improving the efficiency of anti-tumor drug development and the study of drug action mechanisms.

[0008] To solve the above problems, the present invention provides a high-sensitivity detection chip for tumor cells, which sequentially comprises a control membrane, a chip membrane and an electrode plate from top to bottom;

[0009] The chip membrane includes: a first flexible membrane with biocompatibility, a first channel provided on the first flexible membrane and penetrating through the upper and lower ends for accommodating liquid, a plurality of culture grooves provided on the first flexible membrane for culturing single tumor cells, and a plurality of flow grooves provided on the first flexible membrane for transporting the liquid in the first channel to each culture groove; a limiting member for limiting a single tumor cell is arranged in each culture groove; a second channel penetrating through the upper and lower ends of the first flexible membrane for discharging or injecting liquid is opened at one end of each culture groove away from the flow groove.

[0010] The control membrane includes: a second flexible membrane with biocompatibility, a third channel provided on the second flexible membrane and penetrating through the upper and lower ends for discharging or injecting liquid, a plurality of control structures provided on the second flexible membrane for controlling the on-off of the flow grooves, and a plurality of fourth channels provided on the second flexible membrane and penetrating through the upper and lower ends for discharging or injecting liquid; the third channel is communicated with the first channel; the number of the fourth channels is the same as that of the second channels, and each fourth channel is communicated with the corresponding second channel; the number of the control structures is the same as that of the culture grooves, and each control structure includes an air flow groove provided on the second flexible membrane and two air ports provided at both ends of the air flow groove and communicated with the air flow groove, and the air flow groove is located above the corresponding flow groove; when gas enters the air port, the air flow groove expands and squeezes the flow groove, so that the flow groove is closed; when gas is discharged from the air port, the air flow groove contracts, so that the flow groove is unblocked.

[0011] A three - electrode system with a quantity corresponding one - to - one to the quantity of culture tanks is arranged on the electrode plate. The three - electrode system is used to detect the change in liquid current in the corresponding culture tank. Each three - electrode system includes a working electrode, a counter electrode, and a reference electrode. One end of the working electrode, the counter electrode, and the reference electrode in each three - electrode system is located in the culture tank for contacting the liquid in the culture tank, and the other end is used for electrical connection with an external computer.

[0012] The beneficial effect of the present invention is that during use, a tumor cell suspension is injected into the third channel on the control membrane, so that the tumor cell suspension flows through the first channel and the flow channel in sequence and then enters each culture tank. The limiting member can intercept the flowing tumor cells, and the limiting member can only accommodate a single tumor cell. After a single tumor cell is intercepted in each limiting member, a culture solution is injected into the third channel on the control membrane, so that the culture solution flows through the first channel and the flow channel in sequence and then enters each culture tank, acting on the single tumor cell in each limiting member. The change in liquid current in the corresponding culture tank is detected by the three - electrode system, and then the concentration changes of superoxide anions and hydrogen peroxide in the culture tank are obtained according to the current change, so as to realize highly sensitive ROS level analysis. In addition, when gas is introduced through the gas port, the air flow channel forms an expansion to squeeze the flow channel, so that all flow channels are closed. When the gas port is opened, the gas in the air flow channel is discharged, and the flow channel is unblocked. Relative to the chip membrane and the control membrane cooperating with each other to form a valve that can control the on - off of liquid, it is used to control the inflow and stop of liquid in different culture tanks, making the detection more accurate.

[0013] Further, the limiting member is in a C - shape, and the opening of the limiting member faces the liquid outlet at the connection of the flow channel and the culture tank.

[0014] The beneficial effect of this setting is that the C - shaped limiting member can intercept the tumor cells flowing out of the flow channel.

[0015] Further, the electrode plate includes a transparent glass substrate.

[0016] The beneficial effect of this setting is that the transparent glass substrate can facilitate the observation by the staff, and the rigidity of the glass substrate can provide effective support for the control membrane and the chip membrane. At the same time, it is convenient to set the three - electrode system on it, and the cost of the glass substrate is relatively low.

[0017] Further, each flow channel has a curved structure. The beneficial effect of this setting is that it can slow down the flow rate of the tumor cell suspension, so that the tumor cells in the tumor cell suspension are not easily squeezed together due to too fast a flow rate, which is not conducive to the limiting member intercepting a single tumor cell.

[0018] Further, the diameter of each flow channel is 30μm; the opening port diameter of each limiting member is 20μm.

[0019] The beneficial effect of this setting is that this size enables the tumor cells in the tumor cell suspension to be arranged one by one along the flow direction as much as possible, facilitating the interception of single tumor cells by the limiting member.

[0020] Further, the production materials of the first flexible film and the second flexible film include polydimethylsiloxane.

[0021] The beneficial effect of this setting is that polydimethylsiloxane materials have very good flexibility and good biocompatibility, and will not have an impact on tumor cells beyond the experiment.

[0022] Further, the production material of the contact surface between the working electrode and the liquid in the culture tank includes a mixture of graphene and platinum metal nanoparticles, the counter electrode is a platinum electrode, and the reference electrode is an Ag / AgCl electrode.

[0023] The beneficial effect of this setting is that graphene is a very good ROS-sensitive material, which can effectively detect the concentration changes of superoxide anions and hydrogen peroxide in the culture tank. Platinum materials have strong inertness and are not easily corroded by liquids. The Ag / AgCl electrode can provide a stable potential.

[0024] Further, the present invention also provides a preparation method for a highly sensitive tumor cell detection chip, which is used to prepare a highly sensitive tumor cell detection chip described in any one of the above claims, and includes the following steps:

[0025] S1. Clean the single-sided polished monocrystalline silicon wafer with a mixed solution of sulfuric acid and hydrogen peroxide, and the volume ratio of sulfuric acid to hydrogen peroxide is 7:1;

[0026] S2. Coat the outer surface of the monocrystalline silicon wafer with photoresist, and perform pre-baking, photolithography, post-baking, development, spin-drying, and hard-baking on the monocrystalline silicon wafer in sequence to obtain two silicon wafer molds. A structure for forming an air flow channel is formed on one silicon wafer mold, and a structure for forming a flow channel and a culture tank is formed on the other silicon wafer mold;

[0027] S3. Incubate the two silicon wafer molds in a fluorosilane vapor box;

[0028] S4. Mix the polydimethylsiloxane liquid and the curing agent liquid to obtain a polydimethylsiloxane mixture. The weight ratio of the polydimethylsiloxane liquid to the curing agent liquid is 10:1. Place the polydimethylsiloxane mixture in a vacuum dryer and let it stand to remove most of the bubbles;

[0029] S5. Pour the polydimethylsiloxane mixture into the two silicon wafer molds;

[0030] S6. Place the two silicon wafer molds in an oven and heat until the polydimethylsiloxane mixture solidifies;

[0031] S7. Demold the cured polymethylsiloxane mixture to obtain a first flexible film and a second flexible film respectively. An air flow groove is formed on the second flexible film, and a flow groove and a culture groove are formed on the first flexible film.

[0032] S8. Use a syringe to create a first channel and a second channel on the first flexible film; use a syringe to create a third channel, a fourth channel and an air port on the second flexible film.

[0033] S9. Attach white films to the upper and lower surfaces of each first flexible film and second flexible film for storage.

[0034] S10. Clean the transparent glass substrate to obtain a transparent glass substrate, and then coat a photoresist on the transparent glass substrate.

[0035] S11. After lithography, deep reactive ion etching, electron beam evaporation and wet photoresist stripping in sequence, sputter a working electrode, a counter electrode and a reference electrode on the transparent glass substrate. Then, after lithography, plasma treatment and acetone photoresist stripping in sequence, form a circuit on the working electrode, the counter electrode and the reference electrode.

[0036] S12. Anneal the transparent glass substrate in an argon environment to obtain an electrode plate.

[0037] S13. Tear off the white films on the first flexible film and the second flexible film, and put the first flexible film, the second flexible film and the transparent glass substrate into a plasma cleaner for cleaning.

[0038] S14. Bond the first flexible film, the second flexible film and the transparent glass substrate together from top to bottom in sequence to obtain a highly sensitive tumor cell detection chip.

[0039] Furthermore, the present invention also provides a method for using a highly sensitive tumor cell detection chip, which is applied to the highly sensitive tumor cell detection chip described in any one of the above, and includes the following steps:

[0040] A1. Inject a tumor cell suspension into the third channel, so that the tumor cell suspension flows through the first channel and the flow groove in sequence and then enters each culture groove.

[0041] A2. Observe the interception situation of the limiting members in the culture groove through a microscope until a single tumor cell is intercepted in each limiting member.

[0042] A3. Stop injecting the tumor cell suspension, and introduce gas into the air port corresponding to the limiting member. After the air flow groove expands, it squeezes the flow groove, so that the flow groove closes to drain the remaining tumor cell suspension.

[0043] A4. Open all air vents to discharge gas. All air flow grooves contract, enabling all flow grooves to form a circulation path. Inject culture medium into the third channel. The culture medium flows through the first channel and the flow grooves in sequence and enters each culture tank, such that individual tumor cells in each culture tank are all affected by the culture medium.

[0044] A5. Detect the current change in the corresponding culture tank through a three - electrode system, and obtain the concentration changes of superoxide anions and hydrogen peroxide in the culture tank based on the current change.

[0045] A6. Introduce gas into all air vents. The air flow grooves expand and squeeze the flow grooves, causing all flow grooves to close. Open the air vent corresponding to the culture tank where the anti - tumor drug liquid needs to be injected, discharge the gas in the air flow groove, making the flow groove unblocked. Inject the anti - tumor drug liquid from the fourth channel corresponding to the culture tank. The anti - tumor drug liquid enters the culture tank through the second channel and acts on individual tumor cells.

[0046] A7. After waiting for the anti - tumor drug liquid to take effect, detect the liquid current change in the culture tank through a three - electrode system, and obtain the concentration changes of superoxide anions and hydrogen peroxide in the culture tank based on the current change. Description of the Drawings

[0047] Figure 1 It is a three - dimensional schematic diagram of the disassembly of the control membrane, chip membrane, and electrode plate in the present invention;

[0048] Figure 2 It is a schematic diagram of the lower surface of the control membrane in the present invention;

[0049] Figure 3 It is a schematic diagram of the lower surface of the chip membrane in the present invention;

[0050] Figure 4 It is a schematic diagram of the lower surface of the electrode plate in the present invention;

[0051] Figure 5 It is a specific schematic diagram of the three - electrode system structure of an embodiment of the electrode plate in the present invention.

[0052] Description of the Reference Numerals:

[0053] 1 - Second flexible membrane, 2 - First flexible membrane, 3 - Transparent glass substrate, 11 - Third channel, 12 - Fourth channel, 13 - Air vent, 14 - Air flow groove, 21 - First channel, 22 - Culture tank, 23 - Flow groove, 31 - Three - electrode system, 221 - Limiting member, 222 - Second channel, 311 - Working electrode, 312 - Counter electrode, 314 - Reference electrode, First working electrode 3111, Second working electrode 3112, First counter electrode 3121, Second counter electrode 3122, First reference electrode 3131, Second reference electrode 3132. Detailed Embodiments

[0054] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following will describe in detail the specific embodiments of the present invention with reference to the accompanying drawings.

[0055] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "left", "right", "front", "rear", etc. is based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of the present invention is usually placed during use. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0056] This embodiment provides a highly sensitive detection chip for tumor cells, which is characterized in that it includes: a control membrane, a chip membrane, and an electrode plate bonded together in sequence from top to bottom;

[0057] The chip membrane includes: a first flexible membrane 2 with biocompatibility, a first channel 21 provided on the first flexible membrane 2 and penetrating through the upper and lower ends for accommodating liquid, a plurality of culture grooves 22 provided on the lower surface of the first flexible membrane 2 for culturing single tumor cells, and a plurality of flow grooves 23 provided on the lower surface of the first flexible membrane 2 for transporting the liquid in the first channel 21 into each culture groove 22; when the chip membrane is bonded to the electrode plate, a culture chamber and a flow channel are respectively formed between the electrode plate and the culture groove 22 and the flow groove 23; a limiting member 221 for limiting single tumor cells is provided in each culture groove 22; a second channel 222 penetrating through the upper and lower ends of the first flexible membrane 2 for discharging or injecting liquid is provided at one end of each culture groove 22 away from the flow groove 23;

[0058] The control membrane includes: a second flexible membrane 1 with biocompatibility, a third channel 11 disposed on the second flexible membrane 1 and penetrating through the upper and lower ends for discharging or injecting liquid, a plurality of control structures disposed on the second flexible membrane 1 for controlling the on / off of the flow channels 23, and a plurality of fourth channels 12 disposed on the second flexible membrane 1 and penetrating through the upper and lower ends for discharging or injecting liquid; the third channel 11 communicates with the first channel 21, and each of the fourth channels 12 communicates with the corresponding second channel 222; the number of the fourth channels 12 is the same as that of the second channels 222; the number of the control structures is the same as that of the culture tanks 22; each control structure includes an air flow channel 14 disposed on the lower surface of the second flexible membrane 1 and two air ports 13 disposed at both ends of the air flow channel 14 and communicating with the air flow channel 14, and the air flow channel 14 is located above the corresponding flow channel 23; when the control membrane is bonded to the chip membrane, an air flow path is formed between the upper surface of the chip membrane and the air flow channel 14; when gas enters the air ports 13, the air flow path expands and squeezes the flow channel 23, so that the flow channel 23 is closed; when the gas is discharged from the air ports 13, the air flow path contracts, so that the flow channel 23 is unblocked;

[0059] A three-electrode system 31 with the same number as that of the culture tanks 22 is disposed on the electrode plate, and the three-electrode system 31 is used for detecting the change of liquid current in the corresponding culture tank 22; each three-electrode system 31 includes a working electrode 311, a counter electrode 312 and a reference electrode 313; one ends of the working electrode 311, the counter electrode 312 and the reference electrode 313 in each three-electrode system 31 are all located in the culture tank 22 for contacting with the liquid in the culture tank 22, and the other ends are all used for being electrically connected to an external computer.

[0060] Specifically, the first channel 21 is opened at the central part of the first flexible membrane 2, the number of the culture tanks 22 is eight, the eight culture tanks 22 are evenly distributed along the circumferential direction of the first flexible membrane 2, the number of the flow channels 23 and the control structures is also eight, and the positions of the eight control structures correspond to the eight culture tanks 22 one by one, that is, when the second flexible membrane 1 is bonded to the first flexible membrane 2, the eight control structures are respectively disposed directly above the corresponding eight culture tanks 22; as shown in the attached Figure 3As shown, there are four flow channels 23. One end of each flow channel 23 is connected to the first channel 21, and the other end is connected to two culture tanks 22 through a bifurcation; the third channel 11 is coaxially arranged with the first channel 21, and the fourth channel 12 is coaxially arranged with the corresponding second channel 222; in use, a tumor cell suspension is injected into the third channel 11 on the second flexible membrane 1, so that the tumor cell suspension flows through the first channel 21 and the flow channels 23 in sequence and then enters each culture tank 22; the limiting member 221 can intercept the flowing tumor cells, and the limiting member 221 can only accommodate a single tumor cell. After a single tumor cell is intercepted in each limiting member 221, a culture solution is injected into the third channel 11 on the second flexible membrane 1, so that the culture solution flows through the first channel 21 and the flow channels 23 in sequence and then enters each culture tank 22, acting on the single tumor cell in each limiting member 221; when the control membrane, the chip membrane and the electrode plate are integrally formed, one ends of the working electrode 311, the counter electrode 312 and the reference electrode 313 in each three-electrode system 31 are located in the corresponding culture tank 22. In this way, as long as there is liquid passing through the culture tank 22, the working electrode 311, the counter electrode 312 and the reference electrode 313 can all come into contact with the liquid; the liquid current change in the corresponding culture tank 22 is detected by the three-electrode system 31, and then the concentration changes of superoxide anions and hydrogen peroxide in the culture tank 22 are obtained according to the current change, so as to realize highly sensitive ROS level analysis. In addition, a gas is introduced through the gas port 13, and the air flow channel 14 forms an expansion to squeeze the liquid outlet at the connection between the flow channel 23 and the corresponding connected culture tank 22, so that all the flow channels 23 are deformed to form a closure. The gas port 13 is opened to discharge the gas in the air flow channel 14, so that the flow channel 23 is reset to form a circulation. Relative to the cooperation between the first flexible membrane 2 and the second flexible membrane 1 to form a valve that can control the on-off of the liquid, it is used to control the inflow and stop of the liquid in different culture tanks 22, making the detection more accurate.

[0061] In a preferred embodiment of the present invention, the limiting member 221 is in a C shape, and the opening of the limiting member 221 faces the liquid outlet at the connection between the flow channel 23 and the culture tank 22.

[0062] Specifically, the C-shaped limiting member 221 can effectively intercept a single tumor cell and is not easy to escape.

[0063] In a preferred embodiment of the present invention, the electrode plate includes a transparent glass substrate 3.

[0064] Specifically, the transparent glass substrate can facilitate the observation by the staff, and the rigidity of the glass substrate can provide effective support for the second flexible membrane 1 and the first flexible membrane 2. At the same time, it is convenient to set the three-electrode system 31 on it, and the cost of the glass substrate is relatively low.

[0065] In a preferred embodiment of the present invention, as shown in the appendix Figure 5As shown, in all the three - electrode systems 31, the working electrode 311 includes a first working electrode 3111 and a second working electrode 3112 in an L - shaped structure. The counter electrode 312 includes a first counter electrode 3121 and a second counter electrode 3121 in an L - shaped structure. The reference electrode 313 includes a first reference electrode 3131 and a reference electrode 3132 in an L - shaped structure. The first working electrode 3111 and the second working electrode 3112 are symmetrically arranged with each other, forming a U - shaped with the opening downward, and there is a gap between the ends of the first working electrode 3111 and the second working electrode 3112 that contact the liquid in the culture tank 22. The first counter electrode 3121 and the second counter electrode 3121 are symmetrically arranged with each other, forming a U - shaped with the opening downward, and there is a gap between the ends of the first counter electrode 3121 and the second counter electrode 3121 that contact the liquid in the culture tank 22. The first reference electrode 3131 and the reference electrode 3132 are symmetrically arranged with each other, forming a U - shaped with the opening facing outward, and there is a gap between the ends of the first reference electrode 3131 and the reference electrode 3132 that contact the liquid in the culture tank 22. Among them, the first counter electrode 3121 and the second counter electrode 3121 are both located within the U - shaped formed by the first working electrode 3111 and the second working electrode 3112. The first reference electrode 3131 and the reference electrode 3132 are both located within the U - shaped formed by the first counter electrode 3121 and the second counter electrode 3121. The culture tank 22 is arranged within the three U - shaped formed by surrounding. In this way, it is not easy to affect the growth of tumor cells, and at the same time, detection can also be carried out.

[0066] In a preferred embodiment of the present invention, each flow channel 23 has a curved structure.

[0067] Specifically, as shown in the attached Figure 3 Each flow channel 23 is formed into a serpentine structure by a plurality of regular curves. The purpose is to slow down the flow rate of the tumor cell suspension, so that the tumor cells in the tumor cell suspension are not easily squeezed together due to too fast a flow rate, which is not conducive to the limiting member 221 intercepting single tumor cells. Therefore, those skilled in the art can flexibly adjust the number of regular curves and the amplitude of the curves according to the actual situation.

[0068] In a preferred embodiment of the present invention, the diameter of each flow channel 23 is 30μm; the opening diameter of each limiting member 221 is 20μm.

[0069] Specifically, a single tumor cell is generally 10μm or more. The 30μm diameter size of the flow channel 23 can enable the tumor cell suspension to flow smoothly, and at the same time, enable the tumor cells in the tumor cell suspension to be arranged one by one along the flow direction as much as possible, not easily squeezed together. The 20μm opening diameter of the limiting member 221 can facilitate the limiting member to intercept single tumor cells.

[0070] In a preferred embodiment of the present invention, the production materials of the first flexible film 2 and the second flexible film 1 include polydimethylsiloxane.

[0071] Specifically, the polydimethylsiloxane material has very good flexibility and good biocompatibility, and will not have an impact on tumor cells beyond the experiment, that is, this material will not cause the direct death of tumor cells, but can support the survival of tumor cells.

[0072] In a preferred embodiment of the present invention, the production material of the contact surface between the working electrode 311 and the liquid in the culture tank 22 includes graphene and platinum metal nanoparticles, the counter electrode 312 is a platinum electrode, and the reference electrode 313 is an Ag / AgCl electrode.

[0073] Specifically, the ratio of graphene to platinum metal nanoparticles is 1:1. Graphene is a very good ROS-sensitive material, the platinum material has strong inertness and is not easily corroded by the liquid, and the Ag / AgCl electrode can provide a stable potential.

[0074] The present invention also provides a preparation method for a highly sensitive tumor cell detection chip for preparing a highly sensitive tumor cell detection chip described in any one of the above embodiments, including the following steps:

[0075] S1. Clean the single-sided polished monocrystalline silicon wafer with a mixed solution of sulfuric acid and hydrogen peroxide, and the volume ratio of sulfuric acid to hydrogen peroxide is 7:1;

[0076] S2. Coat the outer surface of the monocrystalline silicon wafer with photoresist, and perform pre-baking, photolithography, post-baking, development, spin-drying, and hard-baking on the monocrystalline silicon wafer in sequence to obtain two silicon wafer molds. A structure for forming the air flow groove 14 is formed on one silicon wafer mold, and a structure for forming the flow groove 23 and the culture tank 22 is formed on the other silicon wafer mold;

[0077] S3. Incubate the two silicon wafer molds in a fluorosilane steam box;

[0078] S4. Mix the polydimethylsiloxane liquid and the curing agent liquid to obtain a polydimethylsiloxane mixed solution. The weight ratio of the polydimethylsiloxane liquid to the curing agent liquid is 10:1. Place the polydimethylsiloxane mixed solution in a vacuum dryer and let it stand to remove most of the bubbles;

[0079] S5. Pour the polydimethylsiloxane mixed solution into the two silicon wafer molds;

[0080] S6. Put the two silicon wafer molds into an oven and heat until the polydimethylsiloxane mixed solution solidifies to form a polydimethylsiloxane solid;

[0081] S7. Demold the polymethylsiloxane cured products in the two silicon wafer molds to obtain the first flexible film 2 and the second flexible film 1 respectively. An air flow groove 14 is formed on the second flexible film 1, and a flow groove 23 and a culture groove 22 are formed on the first flexible film 2.

[0082] S8. Use a syringe to open a first channel 21 and a second channel 222 on the first flexible film 2; use a syringe to open a third channel 11, a fourth channel 12 and an air port 13 on the second flexible film 1.

[0083] S9. Attach white films to the upper and lower surfaces of each first flexible film 2 and second flexible film 1 for preservation.

[0084] S10. Clean the transparent glass substrate to obtain a transparent glass substrate 3, and then coat a photoresist on the transparent glass substrate 3.

[0085] S11. After lithography, deep reactive ion etching, electron beam evaporation and wet photoresist stripping in sequence, a working electrode 311, a counter electrode 312 and a reference electrode 313 are sputtered on the transparent glass substrate 3. After lithography, plasma treatment and acetone photoresist stripping in sequence, a circuit is formed on the working electrode 311, the counter electrode 312 and the reference electrode 313.

[0086] S12. Anneal the transparent glass substrate 3 in an argon environment to obtain an electrode plate.

[0087] S13. Tear off the white films on the first flexible film 2 and the second flexible film 1, and put the first flexible film 2, the second flexible film 1 and the transparent glass substrate 3 into a plasma cleaner for cleaning.

[0088] S14. Bond the first flexible film 2, the second flexible film 1 and the transparent glass substrate 3 together from top to bottom in sequence to obtain a highly sensitive tumor cell detection chip.

[0089] Specifically, the size of the monocrystalline silicon wafer in step S1 is four inches. The photoresist coated in step S2 is SU-8 photoresist; the pre-baking is at a temperature of 95°C for a duration of 27 minutes; according to different masks, the structures on the second flexible film 1 and the first flexible film 2, the flow channel 23, and the air flow channel 14 are lithographed; the post-baking is at a temperature of 95°C for a duration of 5 minutes; the development time is 5 minutes; the drying treatment is carried out at a speed of 2100r*60s; the hard-baking is at 150°C for a duration of 9 minutes. In step S3, the incubation time is 4 hours to facilitate subsequent demolding and peeling; in step S4, the vacuum degree of the vacuum dryer is 13psi, and the standing time is 30 minutes; in step S6, it is heated in an oven at 80°C for 1 hour; the syringe aperture in step S8 is specially made to open the first channel 21, the second channel 222, the third channel 11, the fourth channel 12, and the air port 13 with appropriate sizes. In step S10, the photoresist is SU-8 photoresist. In step S11, the photoresist needs to be lithographed first so that a first groove penetrating through to the transparent glass substrate 3 appears on the photoresist. At this time, a Cr adhesion layer is sputtered on the bottom of the first groove, and silver is sputtered on the Cr adhesion layer. Then the photoresist is peeled off, and the photoresist is coated on the transparent glass substrate 3 again. At this time, the photoresist needs to cover the silver. Then lithography is carried out on the side away from the silver so that a second groove penetrating through to the transparent glass substrate 3 appears on the photoresist. A Cr adhesion layer is sputtered on the bottom of the second groove, and platinum is sputtered on the Cr adhesion layer. Then the photoresist is peeled off, and the photoresist is coated on the transparent glass substrate 3 again. At this time, the photoresist needs to cover the silver and platinum. Then lithography is carried out in the middle of the silver and platinum so that a third groove penetrating through to the transparent glass substrate 3 appears on the photoresist. A Cr adhesion layer is sputtered in the third groove, and gold is sputtered on the Cr adhesion layer. Finally, the photoresist is peeled off again; at this time, the exposed platinum is the counter electrode. Then the silver is electrochemically chlorinated to obtain the reference electrode, and the graphene material with a ratio of 1:1 obtained by weak oxidation is mixed with platinum metal nanoparticles and coated on the gold surface to obtain the working electrode. In step S12, the cleaning time is 60 seconds. In step S13, the second flexible film 1, the first flexible film 2, and the electrode plate are sequentially bonded together from top to bottom by an alignment bonder according to the alignment marks. Additionally, during use, the wires of an external computer are soldered to each electrode with solder paste.

[0090] The present invention also provides a method for using a highly sensitive tumor cell detection chip, which is applied to a highly sensitive tumor cell detection chip described in any one of the above embodiments, and includes the following steps:

[0091] A1. Inject the tumor cell suspension into the third channel 11 so that the tumor cell suspension sequentially flows through the first channel 21 and the flow channel 23 and then enters each culture tank 22;

[0092] A2. Observe the retention situation of the limiting members 221 in the culture tank 22 through a microscope until each limiting member 221 retains a single tumor cell;

[0093] A3. Stop injecting the tumor cell suspension, and introduce gas into the air ports 13 corresponding to the limiting members 221. After the air flow grooves 14 expand, they squeeze the flow groove 23, causing the flow groove 23 to close to drain the remaining tumor cell suspension;

[0094] A4. Open all the air ports 13 to discharge the gas. All the air flow grooves 14 contract, causing all the flow grooves 23 to form a circulation. Inject the culture medium into the third channel 11. The culture medium flows through the first channel 21 and the flow groove 23 in sequence and enters each culture tank 22, so that the single tumor cells in each culture tank 22 are all affected by the culture medium;

[0095] A5. Detect the current change in the corresponding culture tank 22 through the three-electrode system 31, and obtain the concentration changes of superoxide anions and hydrogen peroxide in the culture tank 22 according to the current change;

[0096] A6. Introduce gas into all the air ports 13. The air flow grooves 14 expand and squeeze the flow groove 23, causing all the flow grooves 23 to close; Open the air port 13 corresponding to the culture tank 22 that needs to inject the anti-tumor drug liquid to discharge the gas in the air flow groove 14, causing the flow groove 23 to be in circulation. Inject the anti-tumor drug liquid from the fourth channel 12 corresponding to the culture tank 22. The anti-tumor drug liquid enters the culture tank 22 through the second channel 222 and acts on the single tumor cells;

[0097] A7. After waiting for the anti-tumor drug liquid to act, detect the liquid current change in the culture tank 22 through the three-electrode system 31, and obtain the concentration changes of superoxide anions and hydrogen peroxide in the culture tank 22 according to the current change.

[0098] It should be noted that obtaining the concentration changes of superoxide anions and hydrogen peroxide in the culture tank 22 according to the current change belongs to the common general knowledge in the art and is implemented through software algorithms in an external computer. This technology is not the focus of this application either, so it will not be elaborated in this application.

[0099] In this application, an example of lung cancer cell A549 is also provided to further illustrate the above-mentioned method for using a highly sensitive detection chip for tumor cells. Specifically: Inject the cell suspension of lung cancer cell A549 into the third channel 11. At this time, the tumor cell suspension flows through the first channel 21 and the flow trough 23 in sequence and then enters each culture trough 22. The user can observe the interception situation of the limiting member 221 in each culture trough 22 through a microscope. If it is observed that a single tumor cell enters a certain limiting member 221, the gas port 13 corresponding to this culture trough 22 can be input with gas to close the flow trough 23 corresponding to this culture trough 22 until a single tumor cell is intercepted in all limiting members 221; or directly wait until a single tumor cell is intercepted in all limiting members 221 and stop injecting the limiting member 221; at this time, gas needs to be input into all gas ports 13 to close all flow troughs 23 so as to drain the tumor cell suspension in the flow trough 23; then add culture medium, open all gas ports 13 to open all flow troughs 23, and inject the culture medium into the third channel 11. At this time, the culture medium flows through the first channel 21 and the flow trough 23 in sequence and then enters each culture trough 22 to provide nutrients for the tumor cells. The liquid current change during the growth of the tumor cells in the corresponding culture trough 22 is detected by the three-electrode system 31, and the concentration changes of superoxide anions and hydrogen peroxide in the culture trough 22 are obtained according to the current change; in this example, the anti-tumor drug for lung cancer cell A549 is phenethyl isothiocyanate, that is, PEITC; at this time, open the gas port 13 corresponding to the culture trough 22 where the anti-tumor drug needs to be injected to make the flow trough 23 corresponding to this culture trough 22 flow through, and inject the phenethyl isothiocyanate medicinal liquid from the fourth channel 12 corresponding to this culture trough 22. The phenethyl isothiocyanate medicinal liquid will enter the limiting member 221 through the second channel 222 and act on a single tumor cell. At the same time, the excess phenethyl isothiocyanate medicinal liquid will flow out through the flow trough 23, the first channel 21, and the third channel 11 in sequence; after the phenethyl isothiocyanate medicinal liquid acts on lung cancer cell A549 for a period of time, the liquid current change during the growth of the tumor cells in the corresponding culture trough 22 is detected by the three-electrode system 31, and the concentration changes of superoxide anions and hydrogen peroxide in the culture trough 22 are obtained according to the current change; at this time, compare the differences in the ROS levels of the tumor cells in each different culture trough 22 to form a comparative experiment; in this example, one of the culture troughs 22 is a control experiment, that is, a fluorescent probe reagent is added to one of the culture troughs 22 by the above-mentioned method of adding the phenethyl isothiocyanate medicinal liquid, and the ROS level of the cells is characterized by observing the fluorescence intensity in this culture trough 22 through a microscope; finally, the user can analyze the effect of the anti-tumor drug and the drug action mechanism through the test results in each culture trough 22.

[0100] Although the present disclosure is disclosed as above, the scope of protection of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will all fall within the scope of protection of the present invention.

Claims

1. A highly sensitive detection chip for tumor cells, characterized in that, Including: A control film, a chip film, and an electrode plate bonded together in sequence from top to bottom; The chip film includes: a first flexible film (2) with biocompatibility, a first channel (21) provided on the first flexible film (2) and penetrating through the upper and lower ends for accommodating liquid, a plurality of culture grooves (22) provided on the lower surface of the first flexible film (2) for culturing single tumor cells, and a plurality of flow grooves (23) provided on the lower surface of the first flexible film (2) for transporting the liquid in the first channel (21) into each of the culture grooves (22); when the chip film is bonded to the electrode plate, a culture chamber and a flow channel are respectively formed between the electrode plate and the culture grooves (22) and the flow grooves (23); a limiting member (221) for limiting the single tumor cell is provided in each of the culture grooves (22); a second channel (222) penetrating through the upper and lower ends of the first flexible film (2) for discharging or injecting liquid is provided at one end of each of the culture grooves (22) away from the flow groove (23); The control film includes: a second flexible film (1) with biocompatibility, a third channel (11) provided on the second flexible film (1) and penetrating through the upper and lower ends for discharging or injecting liquid, a plurality of control structures provided on the second flexible film (1) for controlling the on-off of the flow grooves (23), and a plurality of fourth channels (12) provided on the second flexible film (1) and penetrating through the upper and lower ends for discharging or injecting liquid; the third channel (11) is communicated with the first channel (21), and each of the fourth channels (12) is communicated with the corresponding second channel (222); the number of the fourth channels (12) is the same as the number of the second channels (222); the number of the control structures is the same as the number of the culture grooves (22); each of the control structures includes an air flow groove (14) provided on the lower surface of the second flexible film (1) and two air ports (13) provided at both ends of the air flow groove (14) and communicated with the air flow groove (14), and the air flow groove (14) is located above the corresponding flow groove (23); when the control film is bonded to the chip film, an air flow channel is formed between the upper surface of the chip film and the air flow groove (14); when gas enters the air ports (13), the air flow channel expands and squeezes the flow groove (23), so that the flow groove (23) is closed; when the gas is discharged from the air ports (13), the air flow channel contracts, so that the flow groove (23) is unblocked; A three - electrode system (31) with the same number as the number of the culture tanks (22) is arranged on the electrode plate. The three - electrode system (31) is used to detect the change of liquid current in the corresponding culture tank (22). Each three - electrode system (31) includes a working electrode (311), a counter electrode (312), and a reference electrode (313). One ends of the working electrode (311), the counter electrode (312), and the reference electrode (313) in each three - electrode system (31) are located in the culture tank (22) for contacting the liquid in the culture tank (22), and the other ends are all used for electrically connecting to an external computer.

2. The highly sensitive detection chip for tumor cells according to claim 1, wherein The limiting member (221) is in a C - shape, and the opening of the limiting member (221) faces the liquid outlet at the connection of the flow channel (23) and the culture tank (22).

3. The highly sensitive detection chip for tumor cells according to claim 1 or 2, characterized in that The electrode plate includes a transparent glass substrate (3).

4. The highly sensitive detection chip for tumor cells according to claim 1 or 2, characterized in that, Each of the flow channels (23) has a curved structure.

5. The highly sensitive detection chip for tumor cells according to claim 3, wherein The diameter of each flow channel (23) is 30μm; the opening diameter of each limiting member (221) is 20μm.

6. A highly sensitive detection chip for tumor cells according to claim 1, 2 or 5, characterized in that, The manufacturing materials of the first flexible film (2) and the second flexible film (1) include polydimethylsiloxane.

7. A highly sensitive detection chip for tumor cells according to claim 1, 2 or 5, characterized in that, The manufacturing material of the contact surface between the working electrode (311) and the liquid in the culture tank (22) includes graphene and platinum metal nanoparticles. The counter electrode (312) is a platinum electrode, and the reference electrode (313) is an Ag / AgCl electrode.

8. A preparation method of a highly sensitive detection chip for tumor cells, characterized in that, A method for preparing a highly sensitive tumor cell detection chip according to any one of claims 1 to 7, comprising the following steps: S1. Clean a single - side polished single - crystal silicon wafer with a mixed solution of sulfuric acid and hydrogen peroxide, and the volume ratio of sulfuric acid to hydrogen peroxide is 7:

1. S2. Coating a photoresist on the outer surface of the single - crystal silicon wafer, and then successively performing pre - baking, photolithography, post - baking, developing, spin - drying, and hard - baking on the single - crystal silicon wafer to obtain two silicon wafer molds. A structure for forming an air flow channel (14) is formed on one of the silicon wafer molds, and a structure for forming a flow channel (23) and a culture tank (22) is formed on the other silicon wafer mold. S3. Incubate the two silicon wafer molds in a fluorosilane vapor box. S4. Mix a polydimethylsiloxane liquid and a curing agent liquid to obtain a polydimethylsiloxane mixed solution. The weight ratio of the polydimethylsiloxane liquid to the curing agent liquid is 10:

1. Place the polydimethylsiloxane mixed solution in a vacuum dryer and let it stand to remove most of the bubbles. S5. Pour the polydimethylsiloxane mixed solution into the two silicon wafer molds. S6. Put the two silicon wafer molds into an oven and heat until the polydimethylsiloxane mixed solution solidifies to form a polydimethylsiloxane solidified product. S7. Demold the polydimethylsiloxane solidified products in the two silicon wafer molds to obtain the first flexible film (2) and the second flexible film (1) respectively. An air flow channel (14) is formed on the second flexible film (1), and a flow channel (23) and a culture tank (22) are formed on the first flexible film (2). S8. Open a first channel (21) and a second channel (222) on the first flexible film (2) through a syringe; open a third channel (11), a fourth channel (12) and an air port (13) on the second flexible film (1) through a syringe; S9. Attach white films to the upper and lower surfaces of each of the first flexible film (2) and the second flexible film (1) for preservation; S10. Clean the transparent glass substrate to obtain a transparent glass substrate (3), and then coat a photoresist on the transparent glass substrate (3); S11. After lithography, deep reactive ion etching, electron beam evaporation and wet photoresist removal in sequence, sputter a working electrode (311), a counter electrode (312) and a reference electrode (313) on the transparent glass substrate (3), and then form a circuit on the working electrode (311), the counter electrode (312) and the reference electrode (313) after lithography, plasma treatment and acetone photoresist removal in sequence; S12. Anneal the transparent glass substrate (3) in an argon environment to obtain an electrode plate; S13. Tear off the white films on the first flexible film (2) and the second flexible film (1), and put the first flexible film (2), the second flexible film (1) and the transparent glass substrate (3) into a plasma cleaner for cleaning; S14. Bond the first flexible film (2), the second flexible film (1) and the transparent glass substrate (3) together from top to bottom in sequence to obtain the highly sensitive tumor cell detection chip.

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