A microfluidic electrode array chip and its preparation method

Abstract

The present application provides a microfluidic electrode array chip and its preparation method. First, an air pump is squeezed to prevent the liquid medium from entering the migration channel under the action of air pressure. The cells can only grow in the culture chamber where the culture medium is present, forming a free boundary to be migrated; then the air pump is used to pump air to backfill the culture medium into the migration channel, and the cells begin to migrate. Since the core of the whole process is to change the state of cell migration by the flow of liquid matrix, there is no risk of cell or electrode damage; and, during the migration of cells in the migration channel, a microelectrode array is embedded in the substrate to which the cells are attached. The microelectrode array can record the electrical signals in the cell migration. After analyzing the electrical signals, it can be proved that the cells have obvious directional movement in the migration channel. In addition, the microfluidic electrode array chip of the present application can also be prepared by a low-cost method, so it has a wide range of applications.

Description

Technical Field

[0001] The present application relates to the technical field of microfluidic chips, and in particular to a microfluidic electrode array chip and a preparation method thereof. Background Art

[0002] Cell invasion refers to the ability of cells to migrate from one area to another through the extracellular matrix. Cell invasion occurs in both normal and cancerous cells in response to chemical and mechanical stimuli. Prior to migration to a new location, the extracellular matrix is ​​degraded by intracellular proteases. Cell invasion often occurs in processes such as wound repair, angiogenesis, and inflammatory responses, as well as in abnormal tissue infiltration and tumor metastasis.

[0003] Currently, the determination of cell invasiveness usually revolves around two aspects: the ability of cells to pass through obstacles and the ability to move. For example, in order to verify the relationship between the electrical signals and invasiveness of gliomas, the existing technology commonly used methods for glioma invasiveness determination include time-lapse imaging technology, cell scratch experiments, cell isolation area experiments, Transwell experiments, and microfluidics technology. Among them, time-lapse imaging technology can perform long-term and accurate measurements in vivo or in vitro at the single cell or even subcellular scale, but this method is costly and has a small sample size, and is suitable for observing structural changes during the migration of a small number of cells; the Transwell experiment emphasizes the ability of cells to deform and pass through obstacles, but does not reflect the directional movement of cell clusters; the scratch size of the cell scratch experiment depends on the operation of the experimenter, with large human errors, and electrical stimulation causes greater damage to edge cells; microfluidics technology has the characteristics of precisely controlling the patterned growth of cells, providing a powerful tool for cell migration experiments, but the processing cost of microfluidics equipment is high, and the popularization of experimental protocols has certain difficulties.

[0004] Therefore, the prior art lacks a device that can continuously and non-invasively monitor the electrical potential of moving cells and observe the directional migration of cells. Summary of the Invention

[0005] The purpose of this application is to solve at least one of the above-mentioned technical deficiencies, especially the technical defect that the prior art lacks a device that can continuously and non-invasively monitor the electric potential of moving cells and observe the directional migration of cells.

[0006] The present application provides a microfluidic electrode array chip, the chip comprising a glass substrate, a microelectrode array distributed on the surface of the glass substrate, and a microfluidic structure bonded to the surface of the microelectrode array;

[0007] The microelectrode array includes a plurality of sensing electrodes arranged at even intervals, a reference electrode remote from the end of the sensing electrodes, wires connected to each sensing electrode and the reference electrode, and lead probes connected to the ends of each wire, wherein each lead probe is distributed near the corresponding sensing electrode in a shortest path manner;

[0008] The microfluidic structure includes a culture chamber arranged on one side of a first sensing electrode, a migration channel connected to the culture chamber and extending from the first sensing electrode to the reference electrode, and an air pump sealed to the end of the migration channel. The culture chamber is connected to the atmosphere.

[0009] Optionally, the width and material of each sensing electrode are consistent with the width and material of the corresponding conductive line.

[0010] Optionally, the width of each sensing electrode and the corresponding conductive line is 100 μm.

[0011] Optionally, the sensing electrodes have the same length, and the center distance between two adjacent sensing electrodes is 200 μm.

[0012] Optionally, the width of the migration channel is smaller than the length of each sensing electrode.

[0013] Optionally, the number of the sensing electrodes is consistent with the number of acquisition channels of the peripheral circuit.

[0014] The present application also provides a method for preparing a microfluidic electrode array chip, the method comprising:

[0015] A microelectrode array is formed on a surface of a glass substrate using a mask method, wherein the microelectrode array includes a plurality of sensing electrodes arranged evenly spaced apart, a reference electrode remote from the end of the sensing electrode, wires connected to each sensing electrode and the reference electrode, and lead probes connected to the ends of each wire, wherein each lead probe is distributed near the corresponding sensing electrode in a shortest path manner;

[0016] A microfluidic structure is constructed using a mold method, wherein the microfluidic structure includes a culture chamber and a migration channel connected to the culture chamber and corresponding to a plurality of sensing electrodes in the microelectrode array;

[0017] After aligning the starting point of the migration channel with the first sensing electrode of the microelectrode array, the microfluidic structure is attached to the glass substrate and heated, and an air pump is sealed and connected to the end of the heated migration channel to form a microfluidic electrode array chip.

[0018] Optionally, the forming of a microelectrode array on the surface of a glass substrate using a mask method comprises:

[0019] Drawing a mask pattern corresponding to a preset microelectrode array;

[0020] Using wire cutting technology to cut a mask corresponding to the mask pattern on the stainless steel substrate;

[0021] After aligning the mask with the cleaned glass substrate, the aligned glass substrate is coated using magnetron sputtering technology, and the mask on the coated glass substrate is removed to form a microelectrode array on the surface of the glass substrate.

[0022] Optionally, the microfluidic structure is constructed using a mold method, comprising:

[0023] After mixing the PDMS reagent and the curing agent in a ratio of 10:1 and stirring evenly, the mixture was poured into the brass mold, and the brass mold containing the mixture was sent to a vacuum defoamer for defoaming;

[0024] heating the defoamed mixture in the brass mold, and separating the PDMS solid obtained after heating from the brass mold;

[0025] The PDMS solid is cut and punched to form a culture chamber and migration channels connected to the culture chamber and corresponding to the plurality of sensing electrodes in the microelectrode array on the surface of the PDMS solid, thereby obtaining a microfluidic structure.

[0026] Optionally, before aligning the starting point of the migration channel with the first sensing electrode of the microelectrode array, the method further comprises:

[0027] Oxygen plasma is used to modify the microfluidic structure and the surface of the glass substrate.

[0028] It can be seen from the above technical solutions that the embodiments of the present application have the following advantages:

[0029] The present application provides a microfluidic electrode array chip and a preparation method thereof, wherein the chip comprises a glass substrate, a microelectrode array distributed on the surface of the glass substrate, and a microfluidic structure bonded to the surface of the microelectrode array; the microelectrode array comprises a plurality of sensing electrodes arranged at uniform intervals, a reference electrode away from the end of the sensing electrode, wires connected to each sensing electrode and the reference electrode respectively, and lead-out probes connected to the end of each wire, wherein each lead-out probe is distributed near the corresponding sensing electrode in the shortest path manner, which can not only improve the impedance between the reference electrode and the sensing electrode, thereby improving the insulation of the wire to the ground, but also make the conduction between the sensing electrode and the lead-out probe The lines are distributed in the shortest path as much as possible to avoid circuit redundancy, improve coupling rate, and enhance the sensitivity of the chip; the microfluidic structure of the present application includes a culture chamber arranged on one side of the first sensing electrode, a migration channel extending from the first sensing electrode to the reference electrode connected to the culture chamber, and an air pump sealed and connected to the end of the migration channel, and the culture chamber is connected to the atmosphere; the present application can squeeze the air pump to prevent the liquid medium from entering the migration channel under the action of air pressure, and the cells can only grow in the culture chamber where the culture medium exists, forming a free boundary to be migrated; then the air pump is used to pump air to backfill the culture medium into the migration channel, the culture chamber and the migration channel are connected, and the cells begin to migrate. Since the core of the whole process is to change the state of cell migration by the flow of the liquid matrix, there is no risk of cell or electrode damage; and, during the migration of cells in the migration channel, a microelectrode array is embedded in the substrate to which the cells are attached. The microelectrode array can record the electrical signals in the cell migration. After analyzing the electrical signals, it can be proved that the cells have obvious directional movement in the migration channel. In addition, the microfluidic electrode array chip of the present application can also be prepared by a relatively low-cost method, such as a mask method, and therefore has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0031] Figure 1 A schematic diagram of the structure of a microelectrode array provided in an embodiment of the present application;

[0032] Figure 2 A schematic diagram of the working process of the microfluidic electrode array chip provided in an embodiment of the present application;

[0033] Figure 3 Schematic diagram of the equivalent circuit of cell-electrode coupling provided in an embodiment of the present application;

[0034] Figure 4 This is a schematic structural diagram of the sensing electrode of this application;

[0035] Figure 5 A schematic diagram of the structure of the migration channel and the sensing electrode provided in an embodiment of the present application;

[0036] Figure 6 A schematic diagram of a process for preparing a microfluidic electrode array chip provided in an embodiment of the present application;

[0037] Figure 7 This is a diagram showing the results of testing the microfluidic electrode array chip provided in the embodiments of the present application. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0039] In one embodiment, the present application provides a microfluidic electrode array chip, which includes a glass substrate, a microelectrode array distributed on the surface of the glass substrate, and a microfluidic structure bonded to the surface of the microelectrode array.

[0040] The microelectrode array includes a plurality of sensing electrodes arranged at uniform intervals, a reference electrode away from the end sensing electrodes, wires connected to each sensing electrode and the reference electrode, and lead probes connected to the ends of each wire, wherein each lead probe is distributed near the corresponding sensing electrode in a shortest path manner.

[0041] The microfluidic structure includes a culture chamber arranged on one side of a first sensing electrode, a migration channel connected to the culture chamber and extending from the first sensing electrode to the reference electrode, and an air pump sealed to the end of the migration channel. The culture chamber is connected to the atmosphere.

[0042] In the present embodiment, in order to non-destructively collect the electrophysiological signals of sample cells, the present application can use microelectrode arrays and microfluidic technology to construct a microfluidic electrode array chip, and the microfluidic electrode array chip is a passive microelectrode array, which usually uses glass as a substrate, so as to facilitate the observation of cells during culture. In addition, the microelectrode array technology can also monitor the spatiotemporal distribution of electrophysiological signals of cell clusters in a long-term and low-noise manner without interfering with the movement of sample cells. Therefore, the microfluidic electrode array chip of the present application has the advantages of flexible design and processing and non-invasive cells. Microfluidic technology has the characteristics of accurately controlling the patterned growth of cells, thereby providing a powerful tool for cell migration experiments. After combining the two, the present application can obtain a microfluidic electrode array chip that can monitor the spatiotemporal distribution of electrophysiological signals of cell clusters in a long-term and low-noise manner without interfering with the movement of sample cells, and can also accurately control the patterned growth of cells in the process.

[0043] Specifically, the microfluidic electrode array chip of the present application may include a glass substrate, a microelectrode array distributed on the surface of the glass substrate, and a microfluidic structure bonded to the surface of the microelectrode array; wherein, Figure 1 As shown, Figure 1 A schematic diagram of the structure of a microelectrode array provided in an embodiment of the present application; Figure 1 The microelectrode array of the present application may include a plurality of evenly spaced sensing electrodes, a reference electrode remote from the distal sensing electrodes, wires connected to each sensing electrode and reference electrode, and lead probes connected to the distal ends of each wire. Each lead probe is distributed near the corresponding sensing electrode in a shortest path manner, which facilitates wiring and avoids circuit redundancy. Furthermore, the present application connects the reference electrode to ground and places it as far away from the wires and sensing electrodes as possible, thereby increasing the impedance between the reference electrode and the sensing electrode and improving the insulation of the wires from ground.

[0044] Furthermore, if Figure 2 As shown, Figure 2 Schematic diagram of the working process of the microfluidic electrode array chip provided in the embodiment of the present application; wherein, Figure 2 a- Figure 2 c is the working principle diagram of the microfluidic electrode array chip. Figure 2As shown in a-2c, the microfluidic structure of the present application is bonded to the surface of the microelectrode array and includes a culture chamber arranged on one side of the first sensing electrode, a migration channel connected to the culture chamber and extending from the first sensing electrode to the reference electrode, and an air pump sealed to the end of the migration channel, wherein the culture chamber is connected to the atmosphere, allowing a pipette to directly load cells and culture medium from above. The air pump can change the air pressure in the migration channel. The air pump of the present application is preferably a syringe. When using a syringe as an air pump, the cells and culture medium are first loaded into the culture chamber, and then the syringe is squeezed so that the liquid medium cannot enter the migration channel under the action of air pressure, and the cell monolayer is only established in the culture chamber; then, the syringe is evacuated to allow the culture medium in the culture chamber to enter the migration channel. After the cells migrate into the migration channel, microelectrodes are embedded in the substrate to which they are attached, so that the electrophysiological signals of the cell migration can be recorded.

[0045] Figure 2 d is the design diagram of the fence-shaped microelectrode array; Figure 2 In d, the dark blue part is the glass substrate, the yellow part is the sensing electrode, and the light blue part is the microfluidic structure. During the migration of cells, the electrophysiological signals generated during the migration process can be recorded through the sensing electrodes at the bottom. After collecting the signals, they can be used to study the cell migration characteristics.

[0046] Figure 2 e is a schematic diagram of the process of collecting electrical signals of migrating cells; Figure 1 As can be seen, the present invention arranges multiple sensing electrodes evenly spaced on a glass substrate, enabling long-term, low-noise monitoring of the spatiotemporal distribution of electrophysiological signals from cell clusters without interfering with sample cell movement. Furthermore, the present invention places the reference electrode away from the distal sensing electrode, which not only increases the impedance between the reference and sensing electrodes but also improves the insulation of the wire from ground.

[0047] It is understandable that when the present application collects electrophysiological signals during cell migration through a microfluidic electrode array chip, the circuit between the cells and the electrodes can be regarded as a signal collection circuit. Specifically, Figure 3 As shown, Figure 3 Schematic diagram of an equivalent circuit of cell-electrode coupling provided in an embodiment of the present application; wherein, Figure 3 It contains three figures a, b, and c. Figure 3 a is the double-layer model and equivalent circuit of the polar interface, Figure 3 b is the cell-electrode coupling model and equivalent circuit, Figure 3 c is the simplified circuit of the cell-electrode coupling model. Figure 3As can be seen from a-c, the sample cell (blue) is attached to the sensing electrode (orange), and the culture medium fills the gap between the two, which is usually between 40-100 nm wide. The ground symbol represents the reference electrode in the culture medium away from the cell, and the potential is stable. The final amplifier picks up the potential difference between the sensing electrode and the reference electrode. For the convenience of discussion, the model parameters are explained below: the sample cell of this application can be equivalent to the signal source Vs; the cell membrane is similar to the electrode interface, which can be equivalent to the parallel connection of capacitance (capacitance of the phospholipid bilayer) and resistance (impedance of carrier flow through the ion channel), such as Figure 3 b is divided into Rj, Cj facing the electrode and Rnj, Cnj not facing the electrode; R seal Represents the resistance between the cell-electrode coupling gap and the reference electrode. The voltage picked up by the electrode is equivalent to R seal The potential difference between the two ends; the electrode can be expressed as Re, Ce, Rm, Rs; among them, Re is the leakage resistance of the double-layer structure at the electrode interface, Ce is the interface capacitance of the double-layer structure at the electrode interface, Rm is the resistance of the electrode itself, Rs is the resistance of the solution in the cell-electrode coupling gap, Rg and Cg are the parasitic resistance and capacitance of the guide wire to the ground; Za is the actual input impedance of the amplifier; Vin is the input voltage of the amplifier.

[0048] The present application may define the coupling ratio as the ratio of Vin to Vs, and the goal of the present application is to make the coupling ratio as high as possible. Specifically, Figure 3 b can be further simplified to Figure 3 After c, Rj, Cj, and Rs form Zc'; Re, Ce, and Rm form Ze'; and Rg, Cg, and Za form Za'. The larger the Rseal / Zc' and Za' / Ze' ratios, the higher the coupling ratio. Zc' depends on the cells and culture medium and is relatively fixed. Therefore, increasing the distance between the reference electrode and the sensing electrode can increase Rseal, thereby increasing Rseal / Zc'.

[0049] In the above embodiment, the chip includes a glass substrate, a microelectrode array distributed on the surface of the glass substrate, and a microfluidic structure bonded to the surface of the microelectrode array; the microelectrode array includes a plurality of sensing electrodes arranged at uniform intervals, a reference electrode away from the end of the sensing electrode, wires connected to each sensing electrode and the reference electrode, and lead-out probes connected to the ends of each wire, each lead-out probe being distributed near the corresponding sensing electrode in the shortest path manner, which can not only improve the impedance between the reference electrode and the sensing electrode, thereby improving the insulation of the wire to the ground, but also make the wire between the sensing electrode and the lead-out probe as short a path as possible. Distribution, thereby avoiding circuit redundancy, improving coupling rate, and improving chip sensitivity; the microfluidic structure of the present application includes a culture chamber arranged on one side of the first sensing electrode, a migration channel extending from the first sensing electrode to the reference electrode connected to the culture chamber, and an air pump sealed and connected to the end of the migration channel, and the culture chamber is connected to the atmosphere; the present application can squeeze the air pump to prevent the liquid medium from entering the migration channel under the action of air pressure, and the cells can only grow in the culture chamber where the culture medium is present, forming a free boundary to be migrated; then the air pump is used to pump air to backfill the culture medium into the migration channel, the culture chamber and the migration channel are connected, and the cells begin to migrate. Since the core of the whole process is to change the state of cell migration by the flow of the liquid matrix, there is no risk of cell or electrode damage; and, during the migration of cells in the migration channel, a microelectrode array is embedded in the substrate to which it is attached. The microelectrode array can record the electrical signals in the cell migration. After analyzing the electrical signals, it can be proved that the cells have obvious directional movement in the migration channel. In addition, the microfluidic electrode array chip of the present application can also be prepared by a low-cost method, such as the mask method, so it has a wide range of applications.

[0050] In one embodiment, the width and material of each sensing electrode are consistent with the width and material of the corresponding conductive line, which can reduce the resistance Rm of the electrode itself, thereby reducing Ze' and improving the coupling ratio.

[0051] In one embodiment, the width of each sensing electrode and the corresponding conductive line is 100 μm.

[0052] In this embodiment, compared with the traditional microelectrode array (about 10 microns), the microelectrode used in this application increases the width of the sensing electrode and the wire and sets it to 100 μm while keeping the electrode thickness unchanged (100 nanometers). This can increase the cross-sectional area of ​​the conductor and improve the conductivity.

[0053] In one embodiment, Figure 4 As shown, Figure 4 This is a schematic structural diagram of the sensing electrode of this application; Figure 4In the example, the lengths of the sensing electrodes are the same, and the center distance between two adjacent sensing electrodes is 200 μm, which can increase both the cross-sectional area of ​​the conductor and the conductivity.

[0054] In a specific embodiment, the sensing electrode area of ​​the present application can be composed of 32 micro-fence electrodes, and each sensing electrode is linearly distributed along the migration direction of the cell sample. The electrode is 100 μm wide, 3 mm long, and has a center spacing of 200 μm, covering the entire channel width of 6.4 mm, thereby efficiently collecting electrophysiological signals generated during cell migration.

[0055] In one embodiment, Figure 5 As shown, Figure 5 This is a schematic structural diagram of the migration channel and sensing electrodes provided in an embodiment of the present application; the width of the migration channel is smaller than the length of each sensing electrode.

[0056] In this embodiment, Figure 5 As shown, Figure 5 and Figure 1 、 Figure 2 After combining, it can be seen that the width of the migration channel (yellow part) is set to be smaller than the length of each sensing electrode (red part), so that the sensing electrodes exposed to the culture solution environment have the same length, thereby improving the accuracy of signal acquisition and analysis.

[0057] In one embodiment, the number of the sensing electrodes is consistent with the number of acquisition channels of the peripheral circuit.

[0058] In this embodiment, the number of sensing electrodes in the microelectrode array can be determined based on the number of acquisition channels in the peripheral circuitry. For example, in the back-end circuitry, an intan amplifier module has 32 channels, so the number of front-end electrodes is also set to 32. Of course, other numbers of sensing electrodes can be set, depending on the actual situation. A greater number of sensing electrodes can capture more information, but the number of channels required in the back-end processing circuitry also increases, which in turn increases costs. Therefore, the number of sensing electrodes can be set based on the balance between cost and information output, and this is not a limitation.

[0059] In one embodiment, Figure 6 As shown, Figure 6 A schematic flow chart of a method for preparing a microfluidic electrode array chip provided in an embodiment of the present application; the present application also provides a method for preparing a microfluidic electrode array chip, which may include:

[0060] S110: forming a microelectrode array on the surface of a glass substrate using a mask method.

[0061] S120: Construction of microfluidic structures using the mold method.

[0062] S130: After aligning the starting point of the migration channel with the first sensing electrode of the microelectrode array, the microfluidic structure is bonded to the glass substrate and heated, and an air pump is sealed and connected to the end of the heated migration channel to form a microfluidic electrode array chip.

[0063] In this embodiment, when preparing a microfluidic electrode array chip, a mask method can first be used to form a microelectrode array on the surface of a glass substrate. The microelectrode array includes a plurality of sensing electrodes arranged at uniform intervals, a reference electrode away from the end of the sensing electrode, a wire connected to each sensing electrode and the reference electrode, and a lead probe connected to the end of each wire. Each lead probe is distributed near the corresponding sensing electrode in the shortest path manner, which is convenient for wiring and avoids circuit redundancy. In addition, the present application connects the reference electrode to the ground and arranges the reference electrode at a position as far away from the wire and the sensing electrode as possible, thereby increasing the impedance between the reference electrode and the sensing electrode and improving the insulation of the wire to the ground. In addition, the present application adopts a mask method as a processing scheme for the microelectrode array, which simplifies the lift-off photolithography process used in traditional microelectrode processing, thereby effectively improving the preparation efficiency.

[0064] Furthermore, the present application can also use a mold method to construct a microfluidic structure, which includes a culture chamber and a migration channel connected to the culture chamber and corresponding to multiple sensing electrodes in the microelectrode array. The culture chamber is connected to the atmosphere, allowing a pipette to load cells and culture medium directly from above, and the migration channel can be used for cell migration.

[0065] After the present application prepares the microelectrode array and the microfluidic structure, the present application can also align the starting point of the migration channel with the first sensing electrode of the microelectrode array, and then bond the microfluidic structure to the glass substrate and heat it, and seal the air pump at the end of the heated migration channel to form a microfluidic electrode array chip.

[0066] For example, the present application can align the first sensing electrode of the microelectrode array with the starting point of the migration channel in the microfluidic structure under a microscope, then quickly fit the microfluidic structure and the glass substrate, heat them at 60°C for 10 minutes, and finally use a silicone hose to connect the air pump interface and the end of the migration channel, and seal the interface with glue to obtain the final microfluidic electrode array chip. This microelectrode array can record the electrical signals during cell migration. By analyzing the electrical signals, it can be proved that the cells have obvious directional movement in the migration channel.

[0067] In a specific embodiment, Figure 7 As shown, Figure 7This is a diagram showing the results of testing the microfluidic electrode array chip provided in the embodiment of the present application; wherein, Figure 7 a-7c shows the sealing verification results of the chip after adding ink for 48 hours; Figure 7 d-7f shows the chip fluid control test results; Figure 7 g-7h shows typical fluorescence microscopic images of glioma cells migrating within 24 h; Figure 7 i shows the distribution of cell numbers on each sensing electrode over time. Figure 7 As can be seen from a-7c, in terms of fluid testing, the chip did not leak within 48 hours after the ink was added, indicating that the microfluidic structure was well bonded to the glass substrate. Figure 7 The chip fluid control results of d-7f show that by controlling the air pump, the liquid can be stably maintained at any position in the migration channel. Figure 7 As shown in g-7h, the cells underwent significant migration within 24 h after the sample cells were implanted. At 0 h, the cells stayed in front of the first sensing electrode, and at 24 h, the cells had reached the sixth sensing electrode at the farthest. The cell migration speed was about 50 μm / h. Figure 7 i shows the distribution changes of cell numbers on each electrode over 7 days. The colors represent the cell numbers. The results show that as the culture time increases, the cells gradually occupy the entire migration channel from the proximal end to the distal end.

[0068] The above results prove that the air pressure-based microfluidic system can manipulate the initial migration position of cells, and glioma cells have obvious directional movement in the migration microchannel.

[0069] In one embodiment, forming a microelectrode array on the surface of a glass substrate using a mask method in S110 may include:

[0070] S111: Drawing a mask pattern corresponding to a preset microelectrode array.

[0071] S112: Using wire cutting technology to cut the stainless steel substrate to obtain a mask corresponding to the mask pattern.

[0072] S113: After aligning the mask with the cleaned glass substrate, the aligned glass substrate is coated using a magnetron sputtering technique, and the mask on the coated glass substrate is removed to form a microelectrode array on the surface of the glass substrate.

[0073] In this embodiment, to obtain a microelectrode array with a high coupling ratio, the present application can pre-construct a corresponding microelectrode array. The microelectrode array can include multiple sensing electrodes arranged at uniform intervals, a reference electrode remote from the sensing electrodes at the ends, wires connected to each sensing electrode and the reference electrode, and lead probes connected to the ends of each wire. Each lead probe is distributed near the corresponding sensing electrode in a shortest path manner. This facilitates wiring and avoids circuit redundancy. In addition, the present application connects the reference electrode to ground and arranges the reference electrode as far away from the wires and sensing electrodes as possible, thereby increasing the impedance between the reference electrode and the sensing electrode and improving the insulation of the wires from the ground.

[0074] Next, the present application can draw a mask pattern corresponding to the microelectrode array in AutoCAD or other drawing software, and then use wire cutting technology to cut a mask corresponding to the mask pattern on the stainless steel substrate, wherein the thickness of the stainless steel substrate can be selected according to the actual situation, such as selecting a stainless steel substrate of 0.3 mm or other thickness, which is not limited here. After obtaining the mask corresponding to the microelectrode array, the present application can align the mask with the cleaned glass substrate. After alignment, a homemade card slot made in 3D printing can be selected, and the aligned mask and the cleaned glass substrate are fixed in the card slot to coat the glass substrate. When coating, magnetron sputtering technology can be used for coating. The target material is platinum, the sputtering thickness is about 100 nm, and it is carried out at room temperature with a humidity of less than 60%. After the coating is completed, the mask can be removed, and the microelectrode pattern is formed on the glass substrate.

[0075] In this application, a microelectrode array is formed in a glass substrate by the above method. In terms of morphology, the chip circuit structure is complete and clear; the electrode color and size are uniform, the width and the center spacing of the electrodes are about 100 μm, and there is no deformation or disconnection, which is in line with the design expectations, indicating that the mask method for processing microelectrodes is feasible.

[0076] In one embodiment, the use of a mold method to construct a microfluidic structure in S120 may include:

[0077] S121: After mixing the PDMS reagent and the curing agent in a ratio of 10:1 and stirring evenly, the mixture is poured into a brass mold, and the brass mold containing the mixture is sent to a vacuum defoamer for defoaming.

[0078] S122: heating the defoamed mixture in the brass mold, and separating the PDMS solid obtained after heating from the brass mold.

[0079] S123: Cutting and punching the PDMS solid to form a culture chamber and migration channels connected to the culture chamber and corresponding to the plurality of sensing electrodes in the microelectrode array on the surface of the PDMS solid, thereby obtaining a microfluidic structure.

[0080] In this embodiment, when the mold method is used to construct a microfluidic structure, the present application can first mix the PDMS reagent and the curing agent in a ratio of 10:1 and stir evenly, then pour the mixture into a brass mold, and send the brass mold containing the mixture into a vacuum defoamer for defoaming. The defoamed mixture is heated at 80°C for 30 minutes, and then the PDMS solid is separated from the brass mold, and the PDMS is fixed and cut and punched to form a culture chamber and a migration channel connected to the culture chamber. The length of the migration channel corresponds to the length of the sensing electrode and the reference electrode in the microelectrode array. Therefore, when the microfluidic structure prepared by this method is bonded to the surface of the glass substrate, it can be bonded by aligning the starting point of the migration channel with the first sensing electrode of the microelectrode array, thereby obtaining the final microfluidic electrode array chip.

[0081] In one embodiment, before aligning the starting point of the migration channel with the first sensing electrode of the microelectrode array in S130, the method may further include: using oxygen plasma to modify the microfluidic structure and the surface of the glass substrate, so that the microfluidic structure and the glass substrate surface are more easily bonded, and the microfluidic electrode array chip formed by heating after bonding is not prone to leakage.

[0082] Finally, it should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the process, method, article, or device comprising the element.

[0083] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The various embodiments can be combined as needed, and the same or similar parts can be referenced to each other.

[0084] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present application. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A microfluidic electrode array chip, characterized in that: The chip includes a glass substrate, a microelectrode array distributed on the surface of the glass substrate, and a microfluidic structure bonded to the surface of the microelectrode array; The microelectrode array includes a plurality of sensing electrodes arranged at even intervals, a reference electrode remote from the end of the sensing electrodes, wires connected to each sensing electrode and the reference electrode, and lead probes connected to the ends of each wire, wherein each lead probe is distributed near the corresponding sensing electrode in a shortest path manner; The microfluidic structure includes a culture chamber arranged on one side of a first sensing electrode, a migration channel connected to the culture chamber and extending from the first sensing electrode to the reference electrode, and an air pump sealed to the end of the migration channel. The culture chamber is connected to the atmosphere.

2. The microfluidic electrode array chip according to claim 1, characterized in that: The width and material of each sensing electrode are consistent with the width and material of the corresponding wire.

3. The microfluidic electrode array chip according to claim 2, characterized in that: The width of each sensing electrode and the corresponding wire is 100 μm.

4. The microfluidic electrode array chip according to claim 3, characterized in that: The lengths of the sensing electrodes are the same, and the center distance between two adjacent sensing electrodes is 200 μm.

5. The microfluidic electrode array chip according to claim 1, characterized in that: The width of the migration channel is smaller than the length of each sensing electrode.

6. The microfluidic electrode array chip according to claim 1, characterized in that: The number of the sensing electrodes is consistent with the number of acquisition channels of the peripheral circuit.

7. A method for preparing a microfluidic electrode array chip, characterized in that: The method comprises: A microelectrode array is formed on a surface of a glass substrate using a mask method, wherein the microelectrode array includes a plurality of sensing electrodes arranged evenly spaced apart, a reference electrode remote from the end of the sensing electrode, wires connected to each sensing electrode and the reference electrode, and lead probes connected to the ends of each wire, wherein each lead probe is distributed near the corresponding sensing electrode in a shortest path manner; A microfluidic structure is constructed using a mold method, wherein the microfluidic structure includes a culture chamber and a migration channel connected to the culture chamber and corresponding to a plurality of sensing electrodes in the microelectrode array; After aligning the starting point of the migration channel with the first sensing electrode of the microelectrode array, the microfluidic structure is attached to the glass substrate and heated, and an air pump is sealed and connected to the end of the heated migration channel to form a microfluidic electrode array chip.

8. The method for preparing a microfluidic electrode array chip according to claim 7, wherein: The method of forming a microelectrode array on the surface of a glass substrate using a mask method comprises: Drawing a mask pattern corresponding to a preset microelectrode array; Using wire cutting technology to cut a mask corresponding to the mask pattern on the stainless steel substrate; After aligning the mask with the cleaned glass substrate, the aligned glass substrate is coated using magnetron sputtering technology, and the mask on the coated glass substrate is removed to form a microelectrode array on the surface of the glass substrate.

9. The method for preparing a microfluidic electrode array chip according to claim 7, wherein: The method of constructing a microfluidic structure by using a mold method comprises: After mixing the PDMS reagent and the curing agent in a ratio of 10:1 and stirring evenly, the mixture was poured into the brass mold, and the brass mold containing the mixture was sent to a vacuum defoamer for defoaming; heating the defoamed mixture in the brass mold, and separating the PDMS solid obtained after heating from the brass mold; The PDMS solid is cut and punched to form a culture chamber and migration channels connected to the culture chamber and corresponding to the plurality of sensing electrodes in the microelectrode array on the surface of the PDMS solid, thereby obtaining a microfluidic structure.

10. The method for preparing a microfluidic electrode array chip according to any one of claims 7 to 9, characterized in that: Before aligning the starting point of the migration channel with the first sensing electrode of the microelectrode array, the method further includes: Oxygen plasma is used to modify the microfluidic structure and the surface of the glass substrate.

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