CN117420185A - Tumor cell high-sensitivity detection chip, preparation method and use method - Google Patents
Tumor cell high-sensitivity detection chip, preparation method and use method Download PDFInfo
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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Abstract
The invention provides a tumor cell high-sensitivity detection chip, which comprises a control film, a chip film and an electrode plate; the chip film comprises a plurality of culture tanks and a plurality of launders; the number of the launders is the same as that of the culture tanks, and each culture tank is internally provided with a limiting piece for limiting the single tumor cells; the control film comprises a plurality of control structures for controlling the flow groove to be communicated, each control structure comprises a pair of air ports and an air flow groove, when the air ports enter the air, the air flow grooves are expanded and then squeeze the flow groove, so that the flow groove is closed; when the air port discharges air, the air flow groove contracts, so that the flow groove is in circulation; the electrode plate is provided with a plurality of three-electrode systems for detecting the current change of the liquid in the culture tank; according to the invention, the detection of the ROS level of tumor cells is realized on one chip, the high-sensitivity ROS level analysis is realized on the premise of not damaging the cells, and the efficiency of anti-tumor drug development and drug action mechanism research is improved.
Description
Technical Field
The invention relates to the technical field of biological micro-fluidic chips, in particular to a tumor cell high-sensitivity detection chip, a preparation method and a use method.
Background
Reactive oxygen species (Reactive Oxygen Species, ROS hereinafter) are free radical reactive species consisting essentially of superoxide anions (O2-), hydrogen peroxide (H) 2 O 2 ) Hydroxyl radical (. OH) and singlet oxygen (1O) 2 )。
Compared with normal cells, the tumor cells are in a higher oxidation state and are more easily influenced by exogenous oxidation, and the apoptosis of the tumor cells can be induced by breaking the ROS balance of the tumor cells so as to achieve the purpose of eliminating the tumor cells. Thus, treatment of tumors based on ROS is currently recognized as an effective tumor treatment.
The concentration change of ROS is an important detection index for development of anti-tumor drugs and research on treatment mechanisms, and ROS level detection is carried out on tumor single cells, so that the effect of the anti-tumor drugs on the tumor single cells with atypical properties can be further explored. The existing single-cell ROS detection method such as mass spectrometry, fluorescence analysis technology, capillary electrophoresis separation method and the like can not realize in-situ detection of living cells, or has low sensitivity, or single-channel single-index detection, or relies on a large instrument to cause higher cost, so that the single-living-cell ROS in-situ, dynamic and high-sensitivity detection requirements are difficult to meet.
The electrochemical analysis method has the advantages of simple equipment, low cost, high sensitivity, quick response, no need of marking, easy integration and the like, and is very suitable for online real-time dynamic detection of single-cell free radicals. For example, patent document CN115201309a discloses a hydroxyl radical electrochemical sensor, a molecular imprinting polymer is prepared on the surface of an electrode modified by g-C3N4/MWCNTs-COOH by a bulk polymerization method, and the detection limit can reach 3.2x10-9 mol/L by using a differential pulse voltammetry method. As in patent document CN114527185a, a copper-silver-loaded few-layer graphene-based composite material hydrogen peroxide sensor is disclosed, and is detected by cyclic voltammetry, and has positive initial potential, larger reduction current, higher sensitivity under smaller voltage and excellent sensing performance. The method realizes high-sensitivity detection of active oxygen substances, but does not realize detection of single-cell multiple free radicals, and has low electrode integration level.
Besides the detection method, the micro-fluidic chip has obvious advantages in single-cell in-situ detection because the functional unit size is equivalent to the cell size, and other detection methods are easy to integrate. As in patent document CN114540182a, a microfluidic system for detecting circulating tumor cell secretions based on single cell level is developed, enrichment and detection of tumor single cell secretions are realized through a primary spiral structure and a secondary micro-droplet forming structure, compared with detection of group cell secretions, a new analysis system is provided for cell heterogeneity analysis, but quantitative analysis is lacking, and the requirement of single tumor cell ROS in-situ dynamic high-sensitivity detection is not satisfied.
Disclosure of Invention
The invention solves the problems that: the high-sensitivity detection chip for the tumor cells, the preparation method and the use method can integrate micro-fluidic and electrode systems, realize detection of ROS level of the tumor cells on one chip, realize high-sensitivity ROS level analysis on the premise of not damaging the cells, and greatly improve the efficiency of anti-tumor drug development and drug action mechanism research.
In order to solve the problems, the invention provides a tumor cell high-sensitivity detection chip, which is provided with a control film, a chip film and an electrode plate from top to bottom in sequence;
the chip film includes: the cell culture device comprises a first flexible membrane with biocompatibility, a first pore canal which is arranged on the first flexible membrane and penetrates through the upper end and the lower end of the first flexible membrane for accommodating liquid, a plurality of culture tanks which are arranged on the first flexible membrane and are used for culturing single tumor cells, and a plurality of launders which are arranged on the first flexible membrane and are used for conveying the liquid in the first pore canal into each culture tank; a limiting piece for limiting the single tumor cells is arranged in each culture tank; one end of each culture tank far away from the launder is provided with a second pore canal penetrating through the upper end and the lower end of the first flexible membrane for discharging liquid or injecting liquid;
the control film includes: the device comprises a second flexible membrane with biocompatibility, a third pore canal which is arranged on the second flexible membrane and penetrates through the upper end and the lower end for discharging liquid or injecting liquid, a plurality of control structures which are arranged on the second flexible membrane and are used for controlling the flow grooves to open and close, and a plurality of fourth pore canals which are arranged on the first flexible membrane and penetrate through the upper end and the lower end for discharging liquid or injecting liquid; the third pore canal is communicated with the first pore canal; the number of the fourth pore channels is the same as that of the second pore channels, and the fourth pore channels are communicated with the corresponding second pore channels; the number of the control structures is the same as that of the culture tanks, each control structure comprises an airflow tank arranged on the second flexible membrane and two air ports which are arranged at two ends of the airflow tank and communicated with the airflow tank, and the airflow tank is positioned above the corresponding launder; when the air port enters the air, the air flow groove is expanded and then extrudes the flow groove, so that the flow groove is closed; when the air port discharges air, the air flow groove contracts, so that the flow groove circulates;
the electrode plates are provided with three electrode systems, the number of which is in one-to-one correspondence with the number of the culture tanks, and the three electrode systems are used for detecting the corresponding liquid current changes in the culture tanks; each three-electrode system includes a working electrode, a counter electrode, and a reference electrode; one end of each working electrode, counter electrode and reference electrode in each three-electrode system is positioned in the culture tank and used for contacting with liquid in the culture tank, and the other end is used for being electrically and mechanically connected with the outside.
The invention has the beneficial effects that when in use, the tumor cell suspension is injected into the third pore canal on the control membrane, so that the tumor cell suspension sequentially flows through the first pore canal and the launder and then enters each culture tank; the limiting pieces can be used for intercepting the flowing tumor cells, the limiting pieces can only be used for accommodating single tumor cells, after each limiting piece is used for intercepting single tumor cells, culture fluid is injected into a third pore canal on the control membrane, so that the culture fluid sequentially flows through the first pore canal and the flow grooves and then enters each culture groove, the single tumor cells in each limiting piece are acted on, the three-electrode system is used for detecting the liquid current change in the corresponding culture groove, then the concentration change of superoxide anions and hydrogen peroxide in the culture groove is obtained according to the current change, and therefore high-sensitivity ROS level analysis is achieved.
Further, the limiting piece is C-shaped, and the opening of the limiting piece faces to the liquid outlet at the joint of the launder and the culture tank
The beneficial effect of this setting is. The C-shaped limiting piece can intercept tumor cells flowing out of the flow groove.
Further, the electrode plate includes a transparent glass substrate.
The beneficial effect of this setting is, transparent glass base plate can make things convenient for the staff to observe, and glass base plate's rigidity can provide the effective support to control membrane and chip membrane, conveniently sets up three electrode system above it simultaneously, and glass base plate cost is lower.
Further, each launder is of curvilinear configuration. The beneficial effect of this setting is, can slow down the velocity of flow of tumour cell suspension for tumour cell in the tumour cell suspension is difficult for because the velocity of flow is too fast and extrudees together, is unfavorable for the locating part to hold back single tumour cell.
Further, the diameter of each launder was 30 μm; the aperture of the opening end of each limiting piece is 20 mu m.
The beneficial effect of this setting is, this size can make the tumour cell in the tumour cell suspension arrange one by one along the flow direction as far as possible, makes things convenient for the locating part to hold back single tumour cell.
Further, the first flexible film and the second flexible film are made of a material including polymethylsiloxane.
The beneficial effects of this setting are, the polymethylsiloxane material has very good flexibility, and has fine biocompatibility, can not produce the influence beyond the experiment to the tumour cell.
Further, the contact surface manufacturing material of the working electrode and the liquid in the culture tank comprises a mixture of graphene and platinum metal nano particles, wherein the counter electrode is a platinum electrode, and the reference electrode is an Ag/AgCl electrode.
The beneficial effect of this setting is, the graphite alkene is fine ROS sensitive material, can effectively detect the concentration variation of superoxide anion and hydrogen peroxide in the culture tank, and platinum material has very strong inertia, is difficult to by liquid corrosion, and Ag/AgCl electrode can provide stable electric potential.
Further, the invention also provides a preparation method of the tumor cell high-sensitivity detection chip, which is used for preparing the tumor cell high-sensitivity detection chip according to any one of the above, and comprises the following steps:
s1, cleaning a single-side polished monocrystalline silicon wafer by using a mixed solution of sulfuric acid and hydrogen peroxide, wherein the volume ratio of the sulfuric acid to the hydrogen peroxide is 7:1;
s2, coating photoresist on the outer surface of the monocrystalline silicon wafer, and sequentially performing pre-baking, photoetching, post-baking, developing, spin-drying and hard baking on the monocrystalline silicon wafer to obtain two silicon wafer molds, wherein one silicon wafer mold is provided with a structure formed by a gas supply launder, and the other silicon wafer mold is provided with a structure formed by a gas supply launder and a culture tank;
s3, placing the two silicon wafer molds into a fluorosilane steam box for incubation;
s4, mixing the polymethylsiloxane liquid and the curing agent liquid to obtain a polymethylsiloxane mixed liquid, wherein the weight ratio of the polymethylsiloxane liquid to the curing agent liquid is 10:1, and placing the polymethylsiloxane mixed liquid in a vacuum dryer for standing so as to remove most of bubbles;
s5, casting the polymethylsiloxane mixed solution in two silicon wafer molds;
s6, placing the two silicon wafer molds into an oven for heating until the polymethylsiloxane mixed solution is solidified;
s7, demolding the cured polymethylsiloxane mixed solution to obtain a first flexible film and a second flexible film respectively, wherein 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;
s8, forming a first pore canal and a second pore canal on the first flexible membrane through a needle cylinder; a third pore canal, a fourth pore canal and an air port are formed on the second flexible membrane through a needle cylinder;
s9, adhering white films to the upper and lower surfaces of each first flexible film and each second flexible film for storage;
s10, cleaning a transparent glass substrate to obtain a transparent glass substrate, and coating photoresist on the transparent glass substrate;
s11, sputtering a working electrode, a counter electrode and a reference electrode on a transparent glass substrate after photoetching, deep reactive ion etching, electron beam evaporation and wet photoresist removal in sequence, and forming a circuit on the working electrode, the counter electrode and the reference electrode after photoetching, plasma treatment and acetone photoresist removal in sequence;
s12, annealing the transparent glass substrate in an argon environment to obtain an electrode plate;
s13, tearing off the white films on the first flexible film and the second flexible film, and putting the first flexible film, the second flexible film and the transparent glass substrate into a plasma cleaner for cleaning;
and S14, bonding the first flexible film, the second flexible film and the transparent glass substrate together from top to bottom in sequence to obtain the tumor cell high-sensitivity detection chip.
Furthermore, the invention also provides a using method of the tumor cell high-sensitivity detection chip, which is applied to any one of the tumor cell high-sensitivity detection chips and comprises the following steps:
a1, injecting tumor cell suspension into the third pore canal, so that the tumor cell suspension sequentially flows through the first pore canal and the launder and then enters each culture tank;
a2, observing the interception condition of the limiting parts in the culture tank through a microscope until all the limiting parts intercept single tumor cells;
a3, stopping injecting the tumor cell suspension, introducing gas into the gas port corresponding to the limiting piece, and extruding the launder after the gas flow groove is expanded, so that the launder is closed to drain the residual tumor cell suspension;
a4, opening all air ports to discharge air, and shrinking all air flow grooves to enable all flow grooves to circulate, injecting culture solution into the third pore canal, enabling the culture solution to sequentially flow through the first pore canal and the flow grooves to enter each culture groove, and enabling single tumor cells in each culture groove to be acted by the culture solution;
a5, detecting current changes in the corresponding culture tank through a three-electrode system, and obtaining concentration changes of superoxide anions and hydrogen peroxide in the culture tank according to the current changes;
a6, introducing gas into all the gas ports, and forming an expansion extrusion launder by the airflow groove so as to close all the launders; opening a corresponding air port of a culture tank into which the anti-tumor drug liquid is required to be injected, exhausting the air in the air flow groove, enabling the air flow groove to circulate, injecting the anti-tumor drug liquid into a fourth pore canal corresponding to the culture tank, and enabling the anti-tumor drug liquid to enter the culture tank through a second pore canal to act on single tumor cells;
a7, after the liquid action of the antitumor drug is finished, detecting the current change of the liquid in the culture tank by a three-electrode system, and obtaining the concentration change of superoxide anions and hydrogen peroxide in the culture tank according to the current change.
Drawings
FIG. 1 is a schematic perspective view of a control film, a chip film and an electrode plate according to the present invention;
FIG. 2 is a schematic view of the lower surface of the control film according to the present invention;
FIG. 3 is a schematic view of the lower surface of the chip film according to the present invention;
FIG. 4 is a schematic view of the lower surface of the electrode plate according to the present invention;
fig. 5 is a schematic diagram showing a three-electrode system structure of an embodiment of the electrode plate according to the present invention.
Reference numerals illustrate:
1-second flexible film, 2-first flexible film, 3-transparent glass substrate, 11-third channel, 12-fourth channel, 13-gas port, 14-gas flow channel, 21-first channel, 22-culture tank, 23-flow channel, 31-three electrode system, 221-stopper, 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 Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the description of the present invention, it should be noted that, directions or positional relationships indicated by terms such as "upper", "lower", "left", "right", "front", "rear", etc., are based on directions or positional relationships shown in the drawings, or directions or positional relationships in which the inventive product is conventionally put in use, are merely for convenience of describing the present invention and simplifying the description, and are not indicative or implying that the apparatus or element to be referred to must have a specific direction, be configured and operated in a specific direction, and thus should not be construed as limiting the present invention.
The embodiment provides a tumor cell high sensitivity detects chip, its characterized in that includes: a control film, a chip film and an electrode plate which are sequentially bonded together from top to bottom;
the chip film includes: a first flexible membrane 2 having biocompatibility, a first duct 21 provided on the first flexible membrane 2 and penetrating both upper and lower ends for accommodating a liquid, a plurality of culture tanks 22 provided on a lower surface of the first flexible membrane 2 for culturing individual tumor cells, and a plurality of flow tanks 23 provided on a lower surface of the first flexible membrane 2 for transferring the liquid in the first duct 21 into the respective culture tanks 22; when the chip film is bonded on the electrode plate, a culture cavity and a flow channel are respectively formed between the electrode plate and the culture tank 22 and between the electrode plate and the flow groove 23; a limiting piece 221 for limiting the single tumor cells is arranged in each culture tank 22; one end of each culture tank 22 far away from the launder 23 is provided with a second pore canal 222 penetrating through the upper and lower ends of the first flexible membrane 2 for discharging liquid or injecting liquid;
the control film includes: a second flexible membrane 1 having biocompatibility, a third duct 11 provided on the second flexible membrane 1 and penetrating both upper and lower ends for discharging or injecting a liquid, a plurality of control structures provided on the second flexible membrane 1 for controlling the flow grooves 23 to open and close, and a fourth duct 12 provided on the first flexible membrane 1 and penetrating both upper and lower ends for discharging or injecting a liquid; the third duct 11 communicates with the first duct 21, and the fourth duct 12 communicates with the corresponding second duct 222; the number of fourth cells 12 is the same as the number of second cells 222; the number of control structures is the same as the number of culture tanks 22; each control structure comprises an air flow groove 14 arranged on the lower surface of the second flexible film 1 and two air ports 13 which are arranged at two ends of the air flow groove 14 and are communicated with the air flow groove 14, and the air flow groove 14 is positioned above the corresponding flow groove 23; when the control film is bonded on the chip film, an air flow channel is formed between the upper surface of the chip film and the air flow groove 14; when the gas port 13 enters the gas, the gas flow channel expands and then presses the flow groove 23, so that the flow groove 23 is closed; when the gas is exhausted from the gas port 13, the gas flow channel contracts, so that the launder 23 circulates;
the electrode plates are provided with three electrode systems 31 the same as the culture tanks 22 in number, and the three electrode systems 31 are used for detecting the corresponding liquid current changes in the culture tanks 22; each three-electrode system 31 includes a working electrode 311, a counter electrode 312, and a reference electrode 313; one end of the working electrode 311, the counter electrode 312 and the reference electrode 313 in each three-electrode system 31 is located in the culture tank 22 for contact with the liquid in the culture tank 22, and the other end is electrically and mechanically connected to the outside.
Specifically, the first pore canal 21 is formed in the central part of the first flexible membrane 2, the number of the culture tanks 22 is eight, the eight culture tanks 22 are uniformly distributed along the circumferential direction of the first flexible membrane 2, the number of the launders 23 and the control structures are also eight, the positions of the eight control structures are in one-to-one correspondence with the eight culture tanks 22, namely, when the second flexible membrane 1 and the first flexible membrane 2 are bonded together, the eight control structures are respectively arranged right above the corresponding eight culture tanks 22; as shown in fig. 3, the number of the flow grooves 23 is four, one end of each flow groove 23 is communicated with the first pore canal 21, and the other end is communicated with two culture grooves 22 through bifurcation; the third duct 11 is coaxially arranged with the first duct 21, and the fourth duct 12 is coaxially arranged with the corresponding second duct 222; when in use, the tumor cell suspension is injected into the third pore canal 11 on the second flexible membrane 1, so that the tumor cell suspension sequentially flows through the first pore canal 21 and the launder 23 and then enters each culture tank 22; the limiting pieces 221 can retain the flowing tumor cells, and the limiting pieces 221 can only contain single tumor cells, when each limiting piece 221 retains the single tumor cells, the culture solution is injected into the third pore canal 11 on the second flexible membrane 1, so that the culture solution sequentially flows through the first pore canal 21 and the launder 23 and then enters each culture groove 22 to act on the single tumor cells in each limiting piece 221; when the control film, the chip film and the electrode plate are integrally formed, one end of the working electrode 311, the counter electrode 312 and the reference electrode 313 in each three-electrode system 31 are all positioned in the corresponding culture tank 22, so that the working electrode 311, the counter electrode 312 and the reference electrode 313 can be contacted with the liquid as long as the liquid passes through the culture tank 22; the three-electrode system 31 is used for detecting the current change of the liquid in the corresponding culture tank 22, then the concentration change of superoxide anions and hydrogen peroxide in the culture tank 22 is obtained according to the current change, so that the high-sensitivity ROS level analysis is realized, in addition, the gas is introduced into the gas port 13, the gas flow grooves 14 form the liquid outlet at the joint of the expansion extrusion flow grooves 23 and the corresponding communicated culture tank 22, so that all the flow grooves 23 are deformed to form a closed state, the gas port 13 is opened, the gas in the gas flow grooves 14 is discharged, the flow grooves 23 are reset to form circulation, and a valve capable of controlling the liquid to flow is formed relative to the mutual cooperation between the first flexible film 2 and the second flexible film 1 and used for controlling the inflow and the stopping of the liquid in different culture tanks 22, so that the detection is more accurate.
In the preferred embodiment of the invention, the limiting piece 221 is C-shaped, and the opening of the limiting piece 221 faces the liquid outlet of the connection part of the launder 23 and the culture tank 22.
Specifically, the C-shaped limiting member 221 can effectively trap a single tumor cell, and is not easy to escape.
In a preferred embodiment of the present invention, the electrode plate includes a transparent glass substrate 3.
Specifically, the transparent glass substrate can be easily observed by a worker, and the rigidity of the glass substrate can provide effective support for the second flexible film 1 and the first flexible film 2, while facilitating the provision of the three-electrode system 31 thereon, and the glass substrate is low in cost.
In the preferred embodiment of the present invention, as shown in fig. 5, in all three-electrode systems 31, the working electrode 311 comprises a first working electrode 3111 and a second working electrode 3112 having an L-shaped structure, the counter electrode 312 comprises a first counter electrode 3121 and a second counter electrode 3121 having an L-shaped structure, and the reference electrode 313 comprises a first reference electrode 3131 and a reference electrode 3132 having an L-shaped structure; the first working electrode 3111 and the second working electrode 3112 are symmetrically arranged to form a U-shape with a downward opening, and a gap is arranged between one ends of the first working electrode 3111 and the second working electrode 3112, which are in contact with the liquid in the culture tank 22; the first pair of electrodes 3121 and the second pair of electrodes 3121 are symmetrically arranged to form a U shape with a downward opening, and a gap is arranged between one ends of the first pair of electrodes 3121 and the second pair of electrodes 3121, which are contacted with the liquid in the culture tank 22; the first reference electrode 3131 and the reference electrode 3132 are symmetrically arranged to form a U shape, the U-shaped opening faces outwards, and a gap is arranged between the first reference electrode 3131 and one end of the reference electrode 3132, which is in contact with the liquid in the culture tank 22; wherein, the first counter electrode 3121 and the second counter electrode 3121 are both located in a U-shape formed by surrounding the first working electrode 3111 and the second working electrode 3112, and the first reference electrode 3131 and the reference electrode 3132 are both located in a U-shape formed by surrounding the first counter electrode 3121 and the second counter electrode 3121; the culture tank 22 is arranged in three U-shapes formed around; thus, the growth of tumor cells is not easily affected, and the detection can be performed.
In the preferred embodiment of the invention, each runner 23 is of curvilinear configuration.
Specifically, as shown in fig. 3, each flow groove 23 is formed into a serpentine structure by a plurality of regular curves, so as to slow down the flow rate of the tumor cell suspension, so that the tumor cells in the tumor cell suspension are not easy to squeeze together due to too fast flow rate, which is unfavorable for the limiting member 221 to intercept single tumor cells, so that the number and the curve amplitude of the regular curves can be flexibly adjusted according to practical situations by those skilled in the art.
In the preferred embodiment of the invention, each launder 23 has a diameter of 30 μm; the aperture of the open end of each stopper 221 is 20 μm.
Specifically, the diameter of the launder 23 of the single tumor cell is generally 10 μm or more, and the size of the launder 23 of 30 μm can enable the tumor cell suspension to smoothly circulate, and simultaneously enable the tumor cells in the tumor cell suspension to be arranged one by one along the flowing direction as much as possible, so that the tumor cells are not easy to squeeze together, and the aperture of the opening end of the limiting piece 221 is 20 μm, so that the limiting piece can conveniently capture the single tumor cells.
In a preferred embodiment of the invention, the first flexible film 2 and the second flexible film 1 are made of a material comprising polymethylsiloxane.
Specifically, the polymethylsiloxane material has very good flexibility and biocompatibility, and can not generate influence on tumor cells beyond experiments, namely, the material can not cause direct death of the tumor cells, and can be used for survival of the tumor cells.
In a preferred embodiment of the present invention, the material for making the contact surface between the working electrode 311 and the liquid in the culture tank 22 comprises graphene and platinum nanoparticles, the counter electrode 312 is a platinum electrode, and the reference electrode 313 is an Ag/AgCl electrode.
Specifically, the ratio of graphene to platinum metal nano particles is 1:1, the graphene is a good ROS sensitive material, the platinum material has strong inertia and is not easy to be corroded by liquid, and the Ag/AgCl electrode can provide stable potential.
The invention also provides a preparation method of the tumor cell high-sensitivity detection chip, which is used for preparing the tumor cell high-sensitivity detection chip in any one of the above embodiments, and comprises the following steps:
s1, cleaning a single-side polished monocrystalline silicon wafer by using a mixed solution of sulfuric acid and hydrogen peroxide, wherein the volume ratio of the sulfuric acid to the hydrogen peroxide is 7:1;
s2, coating photoresist on the outer surface of the monocrystalline silicon wafer, and sequentially performing pre-baking, photoetching, post-baking, developing, spin-drying and hard baking on the monocrystalline silicon wafer to obtain two silicon wafer molds, wherein one silicon wafer mold is provided with a structure formed by a gas supply launder 14, and the other silicon wafer mold is provided with a structure formed by a gas supply launder 23 and a culture launder 22;
s3, placing the two silicon wafer molds into a fluorosilane steam box for incubation;
s4, mixing the polymethylsiloxane liquid and the curing agent liquid to obtain a polymethylsiloxane mixed liquid, wherein the weight ratio of the polymethylsiloxane liquid to the curing agent liquid is 10:1, and placing the polymethylsiloxane mixed liquid in a vacuum dryer for standing so as to remove most of bubbles;
s5, casting the polymethylsiloxane mixed solution in two silicon wafer molds;
s6, placing the two silicon wafer molds into an oven for heating until the polymethylsiloxane mixed solution is solidified to form a polymethylsiloxane solidified substance;
s7, demolding the polymethylsiloxane solidified materials in the two silicon wafer molds to obtain a first flexible film 2 and a second flexible film 1 respectively, wherein 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;
s8, forming a first pore channel 21 and a second pore channel 222 on the first flexible membrane 2 through a needle cylinder; a third pore canal 11, a fourth pore canal 12 and an air port 13 are formed on the second flexible membrane 1 through a needle cylinder;
s9, adhering white films to the upper surface and the lower surface of each first flexible film 2 and each second flexible film 1 for storage;
s10, cleaning a transparent glass substrate to obtain a transparent glass substrate 3, and then coating photoresist on the transparent glass substrate 3;
s11, sputtering a working electrode 311, a counter electrode 312 and a reference electrode 313 on a transparent glass substrate 3 after photoetching, deep reactive ion etching, electron beam evaporation and wet photoresist removal in sequence, and forming a circuit on the working electrode 311, the counter electrode 312 and the reference electrode 313 after photoetching, plasma treatment and acetone photoresist removal in sequence;
s12, annealing the transparent glass substrate 3 in an argon environment to obtain an electrode plate;
s13, tearing off the white films on the first flexible film 2 and the second flexible film 1, and putting the first flexible film 2, the second flexible film 1 and the transparent glass substrate 3 into a plasma cleaner for cleaning;
and S14, bonding 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 tumor cell high-sensitivity detection chip.
Specifically, in step S1, the size of the monocrystalline silicon piece is four inches. The photoresist coated in the step S2 is SU-8 photoresist; pre-baking to 95 ℃ for 27 minutes; photoetching the structures on the second flexible film 1 and the first flexible film 2, the structures of the flow grooves 23 and the air flow grooves 14 according to different masks; post-baking to 95 ℃ for 5 minutes; development time 5 minutes; spin-drying by a speed of 2100r x 60 s; the hard bake was 150℃for 9 minutes. In the step S3, the incubation time is 4 hours, so that the subsequent demolding and stripping are convenient; the vacuum degree of the vacuum dryer in the step S4 is 13psi, and the standing time is 30 minutes; heating in an oven at 80 ℃ in the step S6 for 1 hour; the syringe aperture in step S8 is specially made for the purpose of forming the first port 21, the second port 222, the third port 11, the fourth port 12 and the air port 13 with the same dimensions. In step S10, the photoresist is SU-8 photoresist. In step S11, the photoresist is subjected to a photolithography process, so that the photoresist presents a first groove penetrating through the transparent glass substrate 3, at this time, a Cr adhesion layer is required to be sputtered at the bottom of the first groove, silver is required to be sputtered on the Cr adhesion layer, then the photoresist is stripped, and the transparent glass substrate 3 is coated with the photoresist again, at this time, the photoresist is required to be covered with silver, then a photolithography process is performed at a side far away from silver, so that the photoresist presents a second groove penetrating through the transparent glass substrate 3, a Cr adhesion layer is sputtered at the bottom of the second groove, platinum is required to be sputtered on the Cr adhesion layer, then the photoresist is stripped, and the transparent glass substrate 3 is coated with the photoresist again, at this time, the photoresist is required to be covered with silver and platinum, then a photolithography process is performed in the middle of silver and platinum, so that the photoresist presents a third groove penetrating through the transparent glass substrate 3, and the Cr adhesion layer is sputtered in the third groove, gold is required to be sputtered on the Cr adhesion layer, and finally the photoresist is stripped; the bare platinum is the counter electrode, then the silver is subjected to electric chlorination treatment to obtain a reference electrode, and graphene material with the ratio of 1:1 obtained by weak oxidation is mixed with platinum metal nano particles 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 bonded together in order from top to bottom by an alignment bonder according to the alignment marks. In addition, in use, wires of an external computer are soldered at the respective electrodes by solder paste.
The invention also provides a using method of the tumor cell high-sensitivity detection chip, which is applied to the tumor cell high-sensitivity detection chip in any one of the above embodiments, and comprises the following steps:
a1, injecting tumor cell suspension into the third pore canal 11, so that the tumor cell suspension sequentially flows through the first pore canal 21 and the launder 23 and then enters each culture tank 22;
a2, observing the interception condition of the limiting pieces 221 in the culture tank 22 through a microscope until all the limiting pieces 221 intercept single tumor cells;
a3, stopping injecting the tumor cell suspension, introducing gas into the gas port 13 corresponding to the limiting piece 221, and extruding the flow groove 23 after the gas flow groove 14 is expanded, so that the flow groove 23 is closed to drain the residual tumor cell suspension;
a4, opening all air ports 13 to discharge air, and shrinking all air flow grooves 14 to enable all flow grooves 23 to circulate, injecting culture solution into the third pore canal 11, enabling the culture solution to sequentially flow through the first pore canal 21 and the flow grooves 23 and enter each culture groove 22, and enabling single tumor cells in each culture groove 22 to be acted by the culture solution;
a5, detecting the current change in the corresponding culture tank 22 through the three-electrode system 31, and obtaining the concentration change of superoxide anions and hydrogen peroxide in the culture tank 22 according to the current change;
a6, introducing gas into all the gas ports 13, and forming expansion extrusion flow grooves 23 by the gas flow grooves 14 to enable all the flow grooves 23 to be closed; opening the corresponding air port 13 of the culture tank 22 into which the anti-tumor medicine liquid is required to be injected, exhausting the air in the air flow groove 14, enabling the flow groove 23 to circulate, injecting the anti-tumor medicine liquid into the fourth pore canal 12 corresponding to the culture tank 22, and enabling the anti-tumor medicine liquid to enter the culture tank 22 through the second pore canal 222 to act on single tumor cells;
a7, after the liquid of the antitumor drug is acted, detecting the current change of the liquid in the culture tank 22 through the three-electrode system 31, and obtaining the concentration change of superoxide anions and hydrogen peroxide in the culture tank 22 according to the current change.
It should be noted that, the concentration change of the superoxide anion and the hydrogen peroxide in the culture tank 22 obtained according to the current change is common knowledge in the art, and is implemented by a software algorithm in an external computer, and the technology is not the focus of the present application, so that the description is not repeated in the present application.
In this application, an embodiment of a lung cancer cell a549 is further provided to further illustrate the use method of the tumor cell high-sensitivity detection chip, specifically: injecting the cell suspension of lung cancer cell A549 into the third pore canal 11, wherein the tumor cell suspension sequentially flows through the first pore canal 21 and the flow groove 23 and then enters each culture groove 22, a user can observe the interception condition of the limiting piece 221 in each culture groove 22 through a microscope, and if a single tumor cell is observed to enter one limiting piece 221, the gas is input into the corresponding gas port 13 of the culture groove 22, so that the flow groove 23 corresponding to the culture groove 22 is closed until all the limiting pieces 221 intercept the single tumor cell; or directly waiting until all the limiting pieces 221 are trapped in a single tumor cell, and stopping injecting the tumor limiting pieces 221; at this time, all the air ports 13 need to be filled with air, so that all the flow grooves 23 are closed, and the tumor cell suspension in the flow grooves 23 is exhausted; then adding a culture solution, opening all air ports 13 to open all flow grooves 23, injecting the culture solution into the third pore canal 11, enabling the culture solution to sequentially flow through the first pore canal 21 and the flow grooves 23 and then enter each culture groove 22 to provide nutrition for tumor cells, detecting the liquid current change in the growth process of the tumor cells in the corresponding culture groove 22 through a three-electrode system 31, and obtaining the concentration change of superoxide anions and hydrogen peroxide in the culture groove 22 according to the current change; in the embodiment, the antitumor drug of lung cancer cell A549 is phenethyl isothiocyanate, namely PEITC; at this time, the air port 13 corresponding to the culture tank 22 into which the antitumor drug is required to be injected is opened, so that the flow groove 23 corresponding to the culture tank 22 flows, the phenethyl isothiocyanate liquid medicine is injected into the phenethyl isothiocyanate liquid medicine from the fourth pore canal 12 corresponding to the culture tank 22, the phenethyl isothiocyanate liquid medicine enters the limiting piece 221 through the second pore canal 222 to act on single tumor cells, and meanwhile, the redundant phenethyl isothiocyanate liquid medicine flows out sequentially through the flow groove 23, the first pore canal 21 and the third pore canal 11; after the phenethyl isothiocyanate liquid medicine acts on lung cancer cells A549 for a period of time, detecting liquid current change in the growth process of tumor cells in the corresponding culture tank 22 through a three-electrode system 31, and obtaining concentration change of superoxide anions and hydrogen peroxide in the culture tank 22 according to the current change; at this time, the difference in ROS levels of tumor cells in each of the different culture tanks 22 was compared, forming a comparative experiment; in this embodiment, one of the culture tanks 22 is a control experiment, i.e., a fluorescent probe reagent is added into one of the culture tanks 22 by adding phenethyl isothiocyanate liquid medicine, and the ROS level of the cells is represented by observing the fluorescence intensity in the culture tank 22 through a microscope; finally, the user can analyze the effect and the mechanism of action of the antitumor drug through the test results in each culture tank 22.
Although the present disclosure is described above, the scope of protection of the present disclosure is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the scope of the invention.
Claims (9)
1. A tumor cell high sensitivity detection chip, characterized by comprising: a control film, a chip film and an electrode plate which are sequentially bonded together from top to bottom;
the chip film includes: a first flexible membrane (2) having biocompatibility, a first duct (21) provided on the first flexible membrane (2) and penetrating both upper and lower ends for accommodating a liquid, a plurality of culture tanks (22) provided on a lower surface of the first flexible membrane (2) for culturing individual tumor cells, and a plurality of flow tanks (23) provided on a lower surface of the first flexible membrane (2) for transferring the liquid in the first duct (21) into each of the culture tanks (22); when the chip film is bonded on the electrode plate, a culture cavity and a flow channel are respectively formed between the electrode plate and the culture tank (22) and between the electrode plate and the flow channel (23); a limiting piece (221) for limiting the single tumor cells is arranged in each culture tank (22); one end of each culture tank (22) far away from the launder (23) is provided with a second pore canal (222) penetrating through the upper end and the lower end of the first flexible membrane (2) for discharging liquid or injecting liquid;
the control film includes: a second flexible membrane (1) with biocompatibility, a third pore canal (11) which is arranged on the second flexible membrane (1) and penetrates through the upper end and the lower end for discharging liquid or injecting liquid, a plurality of control structures which are arranged on the second flexible membrane (1) and are used for controlling the flow groove (23) to open and close, and a fourth pore canal (12) which is arranged on the first flexible membrane (1) and penetrates through the upper end and the lower end for discharging liquid or injecting liquid; the third pore canal (11) is communicated with the first pore canal (21), and the fourth pore canal (12) is communicated with the corresponding second pore canal (222); the number of the fourth pore channels (12) is the same as the number of the second pore channels (222); the number of the control structures is the same as the number of the culture tanks (22); each control structure comprises an airflow groove (14) arranged on the lower surface of the second flexible film (1) and two air ports (13) arranged at two ends of the airflow groove (14) and communicated with the airflow groove (14), and the airflow groove (14) is positioned above the corresponding launder (23); when the control film is bonded on the chip film, an air flow channel is formed between the upper surface of the chip film and the air flow groove (14); when the gas port (13) enters gas, the gas flow channel expands and then presses the launder (23) so that the launder (23) is closed; when the gas is exhausted from the gas port (13), the gas flow channel is contracted, so that the launder (23) circulates;
the electrode plates are provided with three electrode systems (31) the same in number as the culture tanks (22), and the three electrode systems (31) are used for detecting the corresponding liquid current changes in the culture tanks (22); each of the three-electrode systems (31) comprises a working electrode (311), a counter electrode (312) and a reference electrode (313); one end of the working electrode (311), the counter electrode (312) and the reference electrode (313) in each three-electrode system (31) is positioned in the culture tank (22) for contacting with the liquid in the culture tank (22), and the other end is electrically and mechanically connected with the outside.
2. The high-sensitivity tumor cell detection chip according to claim 1, wherein the limiting piece (221) is in a shape of a C, and an opening of the limiting piece (221) faces to a liquid outlet at a connection part of the flow groove (23) and the culture groove (22).
3. The tumor cell high-sensitivity detection chip according to claim 1 or 2, wherein the electrode plate comprises a transparent glass substrate (3).
4. A tumor cell high sensitivity detection chip according to claim 1 or 2, wherein each of the flow cells (23) has a curved structure.
5. A tumor cell highly sensitive detection chip according to claim 3, characterized in that the diameter of each of the flow grooves (23) is 30 μm; the aperture of the opening end of each limiting piece (221) is 20 mu m.
6. The tumor cell high-sensitivity detection chip according to claim 1, 2 or 5, wherein the first flexible membrane (2) and the second flexible membrane (1) are made of a material comprising polymethylsiloxane.
7. The tumor cell high-sensitivity detection chip according to claim 1, 2 or 5, wherein the contact surface manufacturing material of the working electrode (311) and the liquid in the culture tank (22) comprises graphene and platinum metal nano particles, the counter electrode (312) is a platinum electrode, and the reference electrode (313) is an Ag/AgCl electrode.
8. A method for preparing a tumor cell high-sensitivity detection chip, which is used for preparing the tumor cell high-sensitivity detection chip according to any one of claims 1 to 7, and comprises the following steps:
s1, cleaning a single-side polished monocrystalline silicon wafer by using a mixed solution of sulfuric acid and hydrogen peroxide, wherein the volume ratio of the sulfuric acid to the hydrogen peroxide is 7:1;
s2, coating photoresist on the outer surface of the monocrystalline silicon wafer, and sequentially performing pre-baking, photoetching, post-baking, developing, spin-drying and hard baking on the monocrystalline silicon wafer to obtain two silicon wafer molds, wherein one silicon wafer mold is provided with a structure formed by a gas supply launder (14), and the other silicon wafer mold is provided with a structure formed by a gas supply launder (23) and a culture bath (22);
s3, placing the two silicon wafer molds into a fluorosilane steam box for incubation;
s4, mixing the polymethylsiloxane liquid and the curing agent liquid to obtain a polymethylsiloxane mixed liquid, wherein the weight ratio of the polymethylsiloxane liquid to the curing agent liquid is 10:1, and placing the polymethylsiloxane mixed liquid in a vacuum dryer for standing so as to remove most of bubbles;
s5, pouring the polymethylsiloxane mixed solution in two silicon wafer molds;
s6, placing the two silicon wafer molds into an oven for heating until the polymethylsiloxane mixed solution is solidified to form a polymethylsiloxane solidified substance;
s7, demolding the polymethylsiloxane solidified materials in the two silicon wafer molds to obtain a first flexible film (2) and a second flexible film (1), wherein an airflow groove (14) is formed in the second flexible film (1), and a flow groove (23) and a culture groove (22) are formed in the first flexible film (2);
s8, a first pore channel (21) and a second pore channel (222) are formed in the first flexible membrane (2) through a needle cylinder; a third pore canal (11), a fourth pore canal (12) and an air port (13) are formed on the second flexible membrane (1) through a needle cylinder;
s9, attaching white films to the upper and lower surfaces of each first flexible film (2) and each second flexible film (1) for storage;
s10, cleaning a transparent glass substrate to obtain a transparent glass substrate (3), and then coating photoresist on the transparent glass substrate (3);
s11, sputtering a working electrode (311), a counter electrode (312) and a reference electrode (313) on the transparent glass substrate (3) after photoetching, deep reactive ion etching, electron beam evaporation and wet photoresist removal in sequence, and forming a circuit on the working electrode (311), the counter electrode (312) and the reference electrode (313) after photoetching, plasma treatment and acetone photoresist removal in sequence;
s12, annealing the transparent glass substrate (3) in an argon environment to obtain an electrode plate;
s13, tearing off the white films on the first flexible film (2) and the second flexible film (1), and putting the first flexible film (2), the second flexible film (1) and the transparent glass substrate (3) into a plasma cleaner for cleaning;
s14, bonding 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 tumor cell high-sensitivity detection chip.
9. A method for using a tumor cell high-sensitivity detection chip, which is characterized by being applied to the tumor cell high-sensitivity detection chip according to any one of claims 1 to 7, and comprising the following steps:
a1, injecting tumor cell suspension into the third pore canal (11) so that the tumor cell suspension sequentially flows through the first pore canal (21) and the launder (23) and then enters each culture groove (22);
a2, observing the interception condition of the limiting pieces (221) in the culture tank (22) through a microscope until all the limiting pieces (221) intercept single tumor cells;
a3, stopping injecting the tumor cell suspension, introducing gas into the gas port (13) corresponding to the limiting piece (221), and extruding the launder (23) after the airflow groove (14) is expanded, so that the launder (23) is closed to drain the residual tumor cell suspension;
a4, opening all air ports (13) to discharge air, and enabling all air flow grooves (14) to shrink so as to enable all flow grooves (23) to form circulation, injecting culture solution into the third pore canal (11), and enabling the culture solution to sequentially flow through the first pore canal (21) and the flow grooves (23) to enter each culture groove (22) so as to enable single tumor cells in each culture groove (22) to be acted by the culture solution;
a5, detecting the corresponding current change in the culture tank (22) through the three-electrode system (31), and obtaining the concentration change of superoxide anions and hydrogen peroxide in the culture tank (22) according to the current change;
a6, introducing gas into all the gas ports (13), and forming expansion of the gas flow grooves (14) to squeeze the flow grooves (23) so that all the flow grooves (23) are closed; opening the corresponding air port (13) of the culture tank (22) into which the anti-tumor drug liquid is required to be injected, discharging the air in the air flow tank (14) to enable the flow tank (23) to circulate, injecting the anti-tumor drug liquid into the fourth pore channel (12) corresponding to the culture tank (22), and enabling the anti-tumor drug liquid to enter the culture tank (22) through the second pore channel (222) to act on the single tumor cells;
a7, after the anti-tumor drug liquid is acted, detecting the liquid current change in the culture tank (22) through the three-electrode system (31), and obtaining the concentration change of superoxide anions and hydrogen peroxide in the culture tank (22) according to the current change.
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