Cell electric signal detection chip, preparation method and cell limiting inoculation method

Abstract

The invention provides a cell electric signal detection chip, a preparation method and a cell limiting inoculation method.The cell electric signal detection chip comprises a substrate, an insulating layer, a limiting structure layer and a plurality of pits, the plurality of pits penetrate through the insulating layer and the limiting structure layer, and the adjacent pits are connected through grooves; an electrode contact is arranged in each pit, each pit is provided with a corresponding conductive structure, and the electrode contact arranged in each pit is electrically connected with the conductive structure corresponding to each pit; the conductive structures corresponding to the pits are arranged between the substrate and the insulating layer and are mutually independent; the pits are used for accommodating and limiting single cells, and the grooves are used for limiting synaptic connection between the cells. Growth and connection of the cells are limited through the pits and the grooves, random growth of the cells and synapses is avoided, and the accuracy of neural signal detection of the cells is guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of electrophysiological technologies, and particularly relates to a cell electro-signal detection chip, a preparation method thereof, and a cell limited inoculation method. Background Art

[0002] The analysis of nerve electro-signals is one of the main research contents of current neuroinformatics. By detecting nerve electrophysiological activities, the understanding of the signal processing mechanism of brain organoids can be achieved, which helps to promote the research of brain science.

[0003] In the prior art, the commonly used research device is a multi-electrode array (MEA for short). The MEA is usually a conductive microelectrode chip prepared on an insulating substrate, and each microelectrode is connected to an external contact by a wire. During use, the test cells or tissues are directly cultured on the electrodes, and then the signal amplifier is connected through the external contacts to simultaneously record the electrophysiological signals of multiple cells in parallel. The MEA is suitable for long-term recording on a single culture sample, allowing the study of the response of cells to external stimuli over a long period of time. However, the synaptic connections between in vitro cultured cells are often random, which is very different from the actual in vivo environment, and the connection methods of cells are different each time during in vitro culture. Moreover, the nerve signals of cells are greatly affected by the connection methods of surrounding cells. Therefore, the existing MEA chip experiments have great randomness, which not only makes the experiments unable to have precise repeatability, but also unable to obtain the actual signals of individual cells forming a nerve cell network. Therefore, how to propose a detection chip that can overcome the defects of in vitro cultured nerve cells in the prior art has become an urgent technical problem to be solved. Summary of the Invention

[0004] In view of the problems in the prior art, embodiments of the present invention provide a cell electro-signal detection chip, a preparation method thereof, and a cell limited inoculation method, which can at least partially solve the problems existing in the prior art.

[0005] In a first aspect, the present invention proposes a cell electro-signal detection chip, including a substrate, an insulating layer, a limiting structure layer, and a plurality of pits, wherein:

[0006] The substrate, the insulating layer, and the limiting structure layer are sequentially stacked, the plurality of pits penetrate through the insulating layer and the limiting structure layer, and adjacent pits are connected by grooves; an electrode contact is arranged in each pit, each pit has a corresponding conductive structure, and the electrode contact arranged in each pit is electrically connected to the conductive structure corresponding to each pit; the conductive structures corresponding to each pit are arranged between the substrate and the insulating layer and are independent of each other;

[0007] The pits are used to accommodate and limit single cells, and the grooves are used to limit synaptic connections between cells.

[0008] Further, the plurality of pits are arranged in an array.

[0009] Further, the pits are cylindrical.

[0010] Further, the diameter of the pits is greater than the circumscribed circle diameter of a single cell and less than twice the circumscribed circle diameter of a single cell.

[0011] Further, the width of the groove is less than the circumscribed circle diameter of a single cell.

[0012] Further, the electrode contacts are needle-like structures.

[0013] In a second aspect, the present invention provides a method for preparing a cell electrical signal detection chip, which is applied to the cell electrical signal detection chip described in any one of the above embodiments, and includes:

[0014] Forming a first mask layer on a substrate, the first mask layer including a plurality of openings, and the openings corresponding to the electrode contacts one by one;

[0015] Forming microstructures of a plurality of electrode contacts at the substrate corresponding to the plurality of openings and removing the first mask layer;

[0016] Forming a second mask layer on the substrate;

[0017] Forming a conductive layer and removing the second mask layer to expose the corresponding substrate; wherein, the conductive layer on the substrate constitutes a circuit layer;

[0018] Forming an insulating layer on the circuit layer and the substrate;

[0019] Forming a limiting structure layer on the insulating layer, the limiting structure layer including each groove.

[0020] Further, forming microstructures of a plurality of electrode contacts at the substrate corresponding to the plurality of openings and removing the first mask layer includes:

[0021] Deploying self-assembled microspheres at the substrate corresponding to the plurality of openings;

[0022] Performing dry etching on the self-assembled microspheres at the substrate corresponding to each opening to obtain micro-needle structures of the electrode contacts.

[0023] Further, at least one of the first mask layer, the second mask layer, the insulating layer, and the limiting structure layer is prepared by a micro-nano particle patterning process; the micro-nano particle patterning process includes:

[0024] Assemble micro-nano particles on the first surface of the first object or the second surface of the second object to form a micro-nano particle layer; wherein, a preset transfer pattern is provided on the second surface;

[0025] Stack the first object and the second object so that the micro-nano particle layer is located between the first surface and the second surface;

[0026] Press the first object and the second object, wherein a part of the micro-nano particle layer corresponding to the preset transfer pattern combines with the first object, and another part of the micro-nano particle layer combines with the second object;

[0027] Separate the first object from the second object to obtain a patterned micro-nano particle array on the first object, wherein the pattern of the micro-nano particle array is consistent with the preset transfer pattern;

[0028] Modify the micro-nano particle array to obtain a mask layer that tightly binds to the first object.

[0029] Further, the modifying the micro-nano particle array to obtain a mask layer that tightly binds to the first object includes:

[0030] Use the thermal sintering method to completely melt and re-plasticize the micro-nano particles of the micro-nano particle array to obtain a dense mask layer;

[0031] Use the thermal sintering method to partially melt and re-plasticize the micro-nano particles of the micro-nano particle array to obtain a porous mask layer; or,

[0032] Use the chemical modification method to modify the micro-nano particle array to obtain a mask layer.

[0033] In a third aspect, the present invention provides a method for limiting the inoculation of cells on a cell electrical signal detection chip according to any one of the above embodiments, including:

[0034] Drop a cell suspension on the limiting structure layer;

[0035] Centrifuge the cell electrical signal detection chip so that the cells in the cell suspension settle and enter the pits;

[0036] Stop centrifuging the cell electrical signal detection chip and remove the supernatant on the limiting structure layer.

[0037] Further, use statically placing the cell electrical signal detection chip to replace centrifuging the cell electrical signal detection chip to prevent cell damage.

[0038] The cell electro-signal detection chip, preparation method and cell limited inoculation method provided by the embodiments of the present invention include a substrate, an insulating layer, a limiting structure layer and a plurality of pits. The substrate, the insulating layer and the limiting structure layer are sequentially stacked. The plurality of pits penetrate through the insulating layer and the limiting structure layer, and adjacent pits are connected by grooves; an electrode contact is arranged in each pit, each pit has a corresponding conductive structure, and the electrode contact arranged in each pit is electrically connected to the conductive structure corresponding to each pit; the conductive structures corresponding to each pit are arranged between the substrate and the insulating layer and are independent of each other; the pits are used to accommodate and limit single cells, and the grooves are used to limit synaptic connections between cells. The growth and connection of cells are defined by the pits and grooves, avoiding random growth of cells and synapses, and ensuring the accuracy of cell nerve signal detection. Description of the Drawings

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to these drawings. In the drawings:

[0040] Figure 1A is a schematic cross-sectional structure diagram of the cell electro-signal detection chip provided by the first embodiment of the present invention.

[0041] Figure 1B is a schematic top view structure diagram of the cell electro-signal detection chip provided by the first embodiment of the present invention.

[0042] Figure 2 is a schematic diagram of the array arrangement of the pits provided by the second embodiment of the present invention.

[0043] Figure 3 is a schematic flow chart of the preparation method of the cell electro-signal detection chip provided by the third embodiment of the present invention.

[0044] Figure 4 is a schematic flow chart of the preparation method of the cell electro-signal detection chip provided by the fourth embodiment of the present invention.

[0045] Figure 5 is a schematic flow chart of the micro-nano particle patterning process provided by the fifth embodiment of the present invention.

[0046] Figure 6A is a schematic structure diagram after forming the first mask layer on the substrate provided by the sixth embodiment of the present invention.

[0047] Figure 6B is a schematic structure diagram after deploying self-assembled microspheres provided by the sixth embodiment of the present invention.

[0048] Figure 6C It is a schematic structural diagram after forming a microneedle structure on a substrate provided by the sixth embodiment of the present invention.

[0049] Figure 6D It is a schematic structural diagram after forming a second mask layer on a substrate provided by the sixth embodiment of the present invention.

[0050] Figure 6E It is a schematic structural diagram after forming a conductive layer provided by the sixth embodiment of the present invention.

[0051] Figure 6F It is a schematic structural diagram after removing the second mask layer provided by the sixth embodiment of the present invention.

[0052] Figure 6G It is a schematic structural diagram after preparing an insulating layer provided by the sixth embodiment of the present invention.

[0053] Figure 6H It is a schematic structural diagram after preparing a limiting structure layer provided by the seventh embodiment of the present invention.

[0054] Figure 7 It is a schematic flow diagram of a cell limiting inoculation method provided by the seventh embodiment of the present invention. Detailed implementation manners

[0055] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer and more understandable, the following further describes the embodiments of the present invention in detail with reference to the accompanying drawings. Here, the illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but not to limit the present invention. It should be noted that, without conflict, the embodiments and features in the embodiments of this application can be combined with each other arbitrarily. The acquisition, storage, use, processing, etc. of data in the technical solutions of this application all comply with the relevant regulations of laws and regulations. The user information in the embodiments of this application is obtained through legal and compliant channels, and the acquisition, storage, use, processing, etc. of the user information are authorized and consented to by the customers.

[0056] To facilitate the understanding of the technical solutions provided in this application, the relevant content of the technical solutions in this application will be described first.

[0057] To overcome the problems such as random connection of in vitro cultured nerve cells in the prior art, this application proposes a cell electrical signal detection chip with a pit and groove structure. A single cell is cultured in the pit, and the groove realizes the growth of controllable synapses, so as to achieve signal detection with single-cell precision.

[0058] Figure 1A It is a schematic cross-sectional structural diagram of a cell electrical signal detection chip provided by the first embodiment of the present invention, Figure 1BIt is a top - view structural schematic diagram of the cell electrical signal detection chip provided by the first embodiment of the present invention. As Figure 1A and Figure 1B shown, the cell electrical signal detection chip provided by the embodiment of the present invention includes a substrate 1, an insulating layer 2, a limiting structure layer 3, and a plurality of pits 4, where:

[0059] The substrate 1, the insulating layer 2, and the limiting structure layer 3 are stacked in sequence. The plurality of pits 4 penetrate through the insulating layer 2 and the limiting structure layer 3, and adjacent pits 4 are connected by grooves 5; an electrode contact 6 is arranged in each pit 4, each pit 4 has a corresponding conductive structure 7, and the electrode contact 6 arranged in each pit 4 is electrically connected to the conductive structure 7 corresponding to each pit 4; the conductive structures 7 corresponding to each pit 4 are arranged between the substrate 1 and the insulating layer 2 and are independent of each other;

[0060] The pits 4 are used to accommodate and limit a single cell 9, and the grooves 5 are used to limit the synaptic connections 10 between cells 9.

[0061] Specifically, the substrate 1 is used to support the overall chip, and the substrate 1 is made of an insulating material. The insulating layer 2 is used to isolate the conductive structures 7 corresponding to each pit 4. The pits 4 penetrate through the insulating layer 2 and the limiting structure layer 3, can accommodate a single cell, and are used to limit the growth of a single cell; an electrode contact 6 is arranged at the bottom of the pit 4, the electrode contact 6 is electrically connected to the corresponding conductive structure 7, and the electrode contact 6 contacts the cell, and can transmit the electrical signal of the cell through the corresponding conductive structure 7. Adjacent pits 4 are connected by grooves 5, and the grooves 5 can limit the growth of synapses and avoid disordered synaptic connections between cells 9.

[0062] The number of pits 4 and grooves 5 is set according to actual needs, and the embodiment of the present invention does not make a limitation. For example, the number of pits 4 is greater than 64. The arrangement of the pits 4 and grooves 5 is set according to actual needs, and the embodiment of the present invention does not make a limitation. For example, the pits 4 are arranged in an array.

[0063] It can be understood that the size of each pit 4 is larger than the size of a single cell to accommodate a single cell, but the size of the pit 4 cannot accommodate two cells to ensure a one - to - one correspondence between the pits 4 and the cells. The size of the groove 5 allows the growth of synapses between cells, but a single cell cannot pass through the groove 5 to avoid the movement of the cells in the pits 4 through the groove 5, and the groove 5 cannot accommodate a single cell to avoid the appearance of a single cell in the groove 5.

[0064] The cell electro-signal detection chip provided by the embodiment of the present invention includes a substrate, an insulating layer, a limiting structure layer, and a plurality of pits. The substrate, the insulating layer, and the limiting structure layer are stacked in sequence. The plurality of pits penetrate through the insulating layer and the limiting structure layer, and adjacent pits are connected by grooves; an electrode contact is arranged in each pit, each pit has a corresponding conductive structure, and the electrode contact arranged in each pit is electrically connected to the conductive structure corresponding to each pit; the conductive structures corresponding to each pit are arranged between the substrate and the insulating layer and are independent of each other; the pits are used to accommodate and limit single cells, and the grooves are used to limit the synaptic connections between cells. The growth and connection of cells are defined by the pits and grooves, avoiding the random growth of cells and synapses, and ensuring the accuracy of cell nerve signal detection.

[0065] The cell electro-signal detection chip proposed by the embodiment of the present invention can overcome the shortcoming of the random connection of in vitro cultured nerve cells in the prior art and realize the accurate measurement of the electro-signals of single cells in large-scale nerve tissues.

[0066] On the basis of the above embodiments, further, the plurality of pits 4 are arranged in an array.

[0067] For example, as Figure 2 shown, 81 pits 4 are arranged in a 9x9 array, and adjacent pits 4 are connected by grooves 5.

[0068] On the basis of the above embodiments, further, the pit 4 is cylindrical. Since the outer contour of the cell is approximately circular, the pit 4 is set to be cylindrical to facilitate cell growth.

[0069] On the basis of the above embodiments, further, the diameter of the pit 4 is larger than the circumscribed circle diameter of a single cell and smaller than 2 times the circumscribed circle diameter of a single cell.

[0070] Specifically, the diameter of the pit 4 is larger than the circumscribed circle diameter of a single cell to ensure that the pit 4 can accommodate a single cell; the diameter of the pit 4 is smaller than 2 times the circumscribed circle diameter of a single cell, so that the pit 4 corresponds to the cell one by one, avoiding the appearance of multiple cells in the pit 4.

[0071] On the basis of the above embodiments, further, the width of the groove 5 is smaller than the circumscribed circle diameter of a single cell to avoid the appearance of cells in the groove 5.

[0072] On the basis of the above embodiments, further, the electrode contact 6 is a needle-like structure. The electrode contact 6 is set to be a needle-like structure, so that the electrode contact 6 can penetrate into the cell and can more conveniently collect the cell electro-signal.

[0073] Figure 3It is a schematic flowchart of a method for preparing a cell electrical signal detection chip provided by the third embodiment of the present invention. As Figure 3 shown, the method for preparing a cell electrical signal detection chip provided by the embodiments of the present invention, which is applied to the cell electrical signal detection chip described in any of the above embodiments, includes:

[0074] S301. Form a first mask layer on the substrate, where the first mask layer includes a plurality of openings, and the openings correspond to the electrode contacts one by one;

[0075] Specifically, a first mask layer with a first mask pattern can be formed on the substrate. The first mask layer includes a plurality of openings, each opening corresponding to an electrode contact one by one. The substrate is exposed at each opening, and a microstructure of the electrode contact is fabricated at the substrate exposed at each opening.

[0076] Among them, the first mask layer with the first mask pattern can be prepared on the substrate surface through a photolithography process or a micro-nano particle patterning process.

[0077] S302. Form microstructures of a plurality of electrode contacts at the substrate corresponding to the plurality of openings and remove the first mask layer;

[0078] Specifically, at the substrate corresponding to each opening, a microstructure of each electrode contact is prepared through a composite micro-nano manufacturing process based on self-assembled microspheres. The microstructure is conducive to the full contact between the electrode contact and the cell and improves the electrical coupling state with the cell. After forming the microstructures of the plurality of electrode contacts, the first mask layer is removed.

[0079] S303. Form a second mask layer on the substrate;

[0080] Specifically, a second mask layer with a second mask pattern can be formed on the substrate. The substrate covered by the second mask layer will be prepared with an insulating layer subsequently to isolate the conductive structures corresponding to the respective pits.

[0081] For example, the second mask layer with the second mask pattern can be prepared on the substrate surface through a photolithography process or a micro-nano particle patterning process.

[0082] S304. Form a conductive layer and remove the second mask layer to expose the corresponding substrate; wherein, the conductive layer on the substrate constitutes a circuit layer;

[0083] Specifically, a conductive layer is prepared on the substrate not covered by the second mask layer and on the microstructures of the plurality of electrode contacts, and the conductive layer is made of a conductive material. The conductive layer prepared on the substrate not covered by the second mask layer constitutes a circuit layer for transmitting electrical signals; the conductive layer formed on the microstructures of the electrode contacts and the microstructures constitute electrode contacts, and the conductive layer formed on the microstructures is electrically connected to the corresponding circuit layer. After the conductive layer is formed, the second mask layer is removed.

[0084] S305. An insulating layer is formed on the circuit layer and the substrate;

[0085] Specifically, an insulating layer is formed on the circuit layer and the substrate. The insulating layer exposes each electrode contact.

[0086] For example, the insulating layer of the third mask pattern can be prepared on the circuit layer and the substrate through a photolithography process or a micro-nano particle patterning process.

[0087] S306. A limiting structure layer is formed on the insulating layer, and the limiting structure layer includes respective grooves.

[0088] Specifically, a limiting structure layer is formed on the insulating layer, and the limiting structure layer includes respective grooves. The limiting structure layer exposes each electrode contact. The part passing through the insulating layer and the limiting structure layer and exposing the electrode contact forms a pit.

[0089] For example, the limiting structure layer can be prepared on the insulating layer through a photolithography process or a micro-nano particle patterning process.

[0090] The method for preparing a cell electrical signal detection chip provided by an embodiment of the present invention forms a first mask layer on a substrate, the first mask layer includes a plurality of openings, and the openings correspond to the electrode contacts one by one; microstructures of the plurality of electrode contacts are formed at the positions of the substrate corresponding to the plurality of openings and the first mask layer is removed; a second mask layer is formed on the substrate; a conductive layer is formed and the second mask layer is removed to expose the corresponding substrate; wherein, the conductive layer on the substrate constitutes a circuit layer; an insulating layer is formed on the circuit layer and the substrate; a limiting structure layer is formed on the insulating layer, and the limiting structure layer includes respective grooves, realizing the preparation of the cell electrical signal detection chip, providing a new chip for detecting neural signals of cells, and being able to improve the accuracy of detecting neural signals of cells.

[0091] Figure 4 is a schematic flowchart of the method for preparing a cell electrical signal detection chip provided by the fourth embodiment of the present invention. As Figure 4 shown, on the basis of the above embodiments, further, forming the microstructures of the plurality of electrode contacts at the positions of the substrate corresponding to the plurality of openings included in the first mask pattern includes:

[0092] S401. Deploy self-assembled microspheres at the substrate corresponding to the multiple openings;

[0093] Specifically, fill self-assembled microspheres at the substrate corresponding to the multiple openings. A monolayer of microspheres will be self-assembled at the substrate corresponding to each opening, covering the surface of the substrate corresponding to each opening.

[0094] S402. Perform dry etching on the self-assembled microspheres at the substrate corresponding to each opening to obtain the micro-needle structures of the multiple electrode contacts.

[0095] Specifically, by simultaneously etching the self-assembled microspheres and the substrate at the substrate corresponding to each opening through a dry etching process, the micro-needle structures of each electrode contact can be formed at the substrate corresponding to each opening.

[0096] The microspheres play a partial protection role. In the dry etching environment, the etching rate of the part directly in contact with the microspheres is slow, and the etching rate of the gap between the spheres is fast. As time goes by, the micro-needle structures are finally left.

[0097] Based on the above embodiments, further, at least one of the first mask layer, the second mask layer, the insulating layer, and the limiting structure layer is prepared by a micro-nano particle patterning process; as Figure 5 shown, the micro-nano particle patterning process includes:

[0098] S501. Assemble micro-nano particles on the first surface of the first object or the second surface of the second object to form a micro-nano particle layer; wherein, a preset transfer pattern is provided on the second surface;

[0099] Specifically, assemble micro-nano particles on the first surface of the first object or the second surface of the second object to form a micro-nano particle layer. A preset transfer pattern is provided on the second surface. The preset transfer pattern corresponds to a mask pattern, and the mask pattern is the first mask pattern, the second mask pattern, the third mask pattern, or the fourth mask pattern. Among them, the preset transfer pattern is convex or concave, which is selected according to actual needs and is not limited in the embodiments of the present invention. The line width of the micro-nano particle array is in the micron or nano scale, which is selected according to actual needs and is not limited in the embodiments of the present invention. The second object is made of a flexible material, and the flexible material can be selected from one or more of polydimethylsiloxane (PDMS), polyimide (PI), and rubber. The particle size of the micro-nano particles is 100 nm to 100 μm. The surface of the preset transfer pattern can be a discontinuous plane, which is set according to actual needs and is not limited in the embodiments of the present invention.

[0100] When fabricating the first mask layer, the first object is the substrate, the second object is the first template, and a first mask pattern is provided on the first template. When fabricating the second mask layer, the first object is the substrate, the second object is the second template, and a second mask pattern is provided on the second template. When fabricating the insulating layer, the first objects are the circuit layer and the substrate, and the second object is the third template. When fabricating the limiting structure layer, the first object is the insulating layer, and the second object is the fourth template.

[0101] Assemble the micro-nano particles on the one of the first surface and the second surface with a smaller binding force between the micro-nano particles. For example, if the binding force between the micro-nano particles and the first surface of the first object is the first binding force, the binding force between the micro-nano particles and the second surface of the second object is the second binding force, and the first binding force is greater than the second binding force, then assemble the micro-nano particles on the second surface; assemble the micro-nano particles on the second object with a smaller binding force, rather than on the first object with a greater binding force, because the latter will make it difficult for the part of the micro-nano particle layer corresponding to the matching pattern to separate from the first object, and the desired transfer pattern cannot be obtained.

[0102] The material of the micro-nano particles can be selected from one or more of polystyrene, silicon dioxide, and polytetrafluoroethylene (PTFE). Selecting a polymer material with a lower glass transition temperature as the material of the micro-nano particles helps to reduce the heating temperature in subsequent hot pressing and modification processes, further reduce costs, and shorten the preparation time.

[0103] The shape of the micro-nano particles can be spherical, ellipsoidal, etc. When the micro-nano particles are non-spherical particles, the particle size of the micro-nano particles in the embodiments of the present invention refers to the equivalent particle size.

[0104] Regarding the assembly method of the micro-nano particles, the present application does not make any restrictions, as long as it can achieve the assembly of the micro-nano particles on the surface of the first object or the second object. For example, the gas-liquid interface self-assembly process can be used to assemble the micro-nano particles on the second surface of the second object. In this embodiment, the existing gas-liquid interface self-assembly process can be used to assemble the micro-nano particles, so the present application will not elaborate on this.

[0105] S502. Stack the first object and the second object so that the micro-nano particle layer is located between the first surface and the second surface;

[0106] Specifically, oppose the first surface of the first object to the second surface of the second object, stack the first object and the second object, so that the micro-nano particle layer is located between the first surface and the second surface.

[0107] The second object and the first object are stacked in a superposed manner with a micro-nano particle layer located between the first surface and the second surface. In the superposed state, a first part of the micro-nano particle layer corresponding to the convex preset transfer pattern is in contact with both the second surface of the second object and the first surface of the first object, while a second part of the micro-nano particle layer corresponding to the concave mating pattern is only in contact with the second surface of the second object and cannot be in contact with the first surface of the first object. There is a spacing between the second part of the micro-nano particle layer and the first surface of the first object, and this spacing is equal to the height by which the preset transfer pattern protrudes. Since there is a certain binding force between the micro-nano particles and the second object, the second part of the micro-nano particle layer can be held on the second surface of the second object by this binding force without the problem of falling off from the second surface.

[0108] S503. Press the first object and the second object together, wherein a part of the micro-nano particle layer corresponding to the preset transfer pattern binds to the first object, and another part of the micro-nano particle layer binds to the second object;

[0109] Specifically, press the superposed first object and second object together such that a part of the micro-nano particle layer corresponding to the preset transfer pattern binds to the first object, and another part of the micro-nano particle layer, that is, the remaining micro-nano particle layer after removing the part corresponding to the preset transfer pattern, binds to the second object.

[0110] For example, perform thermal pressing on the first object and the second object, wherein the temperature of the thermal pressing is the glass transition temperature of the micro-nano particles. The glass transition temperature of the micro-nano particles is less than 100 °C. Through thermal pressing, the micro-nano particles in the first part of the micro-nano particle layer undergo a glass transition, thereby binding firmly to the first object. In the embodiments of the present invention, existing thermal pressing processes and thermal pressing equipment can be used to perform thermal pressing on the first object and the second object, so this application will not elaborate on this.

[0111] S504. Separate the first object from the second object to obtain a patterned micro-nano particle array on the first object, wherein the pattern of the micro-nano particle array is consistent with the preset transfer pattern;

[0112] Specifically, separate the first object from the second object, and a part of the micro-nano particle layer that binds to the first object forms a patterned micro-nano particle array on the first object, and the pattern of the micro-nano particle array is consistent with the preset transfer pattern.

[0113] Separating the second object from the first object can be removing the second object from the first object or removing the first object from the second object. In either case, the second part of the micro-nano particle layer is removed together with the second object, that is, separated from the entire micro-nano particle layer, while the first part of the micro-nano particle layer is retained on the first object due to its greater binding force with the first object and will not separate from the first object along with the second object. The first part of the micro-nano particle layer remaining on the first surface of the first object is a patterned micro-nano particle array, and the pattern of this array is consistent with the preset transfer pattern, thus realizing the transfer of the preset transfer pattern onto the first object in the form of a micro-nano particle array, with high transfer accuracy, simple process, and low requirements for equipment.

[0114] S505. Modify the micro-nano particle array to obtain a mask layer that tightly binds to the first object.

[0115] Specifically, the thermal sintering method or the chemical modification method can be used to modify the micro-nano particle array to obtain a mask layer that tightly binds to the first object. For the specific modification method, it is selected according to actual needs, and the embodiments of the present invention do not make limitations, as long as it can achieve the tight binding of the micro-nano particle array to the first surface of the first object.

[0116] This application uses micro-nano particles to prepare a mask and uses a second object for patterned transfer. It does not rely on complex optical paths and equipment, has the advantages of low cost, rapid preparation, and low requirements for equipment, and can replace traditional photoresist masks to a certain extent.

[0117] On the basis of the above embodiments, further, the modifying the micro-nano particle array to obtain a mask layer that tightly binds to the first object includes:

[0118] Using the thermal sintering method to completely melt and re-plasticize the micro-nano particles of the micro-nano particle array to obtain a dense mask layer;

[0119] Using the thermal sintering method to partially melt and re-plasticize the micro-nano particles of the micro-nano particle array to obtain a porous mask layer; or,

[0120] Using the chemical modification method to modify the micro-nano particle array to obtain a mask layer.

[0121] For example, an organic solvent can be used to swell the micro-nano particles, and the swollen micro-nano particles fill the gaps between each other and are interconnected to form a mask layer.

[0122] For example, for micro-nano particles made of photosensitive materials, ultraviolet light can be used to irradiate the micro-nano particles to cause them to crosslink and cure, thereby forming a mask layer.

[0123] For example, a first object carrying a micro-nano particle array is placed in a polymer solution. The micro-nano particles serve as growth sites and at the same time as catalysts, causing the chemical substances in the polymer solution to crystallize and grow. The formed crystals fill the gaps between the micro-nano particles and between the micro-nano particles and the first object, thereby forming a dense mask layer. The main component of the micro-nano particles can be a polymer, such as polymers like polystyrene, PP, PVC, etc., or can be an inorganic substance, such as silicon dioxide. The chemical substances in the polymer solution can be the same as the main component of the micro-nano particles, so that the formed crystals can better combine with the micro-nano particles to form a dense mask layer.

[0124] Based on the above embodiments, further, in a direction parallel to the second surface of the second object, the micro-nano particles are closely arranged on the second surface to form a continuous micro-nano particle layer, that is, the micro-nano particles are compactly arranged on the second surface, and any two adjacent micro-nano particles are in contact rather than spaced apart, and the micro-nano particle layer covers the entire preset transfer pattern and the entire mating pattern.

[0125] Based on the above embodiments, further, in a direction perpendicular to the second surface of the second object, the micro-nano particles are arranged in a single layer on the second surface to form a single-layer micro-nano particle layer, that is, only one layer of micro-nano particle layer is arranged on the second surface of the second object, so that all the micro-nano particles can directly contact the second surface.

[0126] Based on the above embodiments, further, the preset transfer pattern is a concave pattern or a convex pattern;

[0127] The assembling of the micro-nano particles on the first surface or the second surface to form a micro-nano particle layer includes:

[0128] The micro-nano particles are assembled on the one of the first surface and the second surface with a smaller binding force between the micro-nano particles.

[0129] Based on the above embodiments, further, the binding force between the first surface and the micro-nano particles is a first binding force, the binding force between the second surface and the micro-nano particles is a second binding force, the first binding force is greater than the second binding force, and the preset transfer pattern is a convex pattern;

[0130] The assembling of the micro-nano particles on the first surface or the second surface to form a micro-nano particle layer includes: The micro-nano particles are assembled on the second surface.

[0131] Based on the above embodiments, further, the binding force between the first surface and the micro-nano particles is the first binding force, the binding force between the second surface and the micro-nano particles is the second binding force, the second binding force is greater than the first binding force, and the preset transfer pattern is a concave pattern;

[0132] The assembling the micro-nano particles on the first surface or the second surface to form a micro-nano particle layer includes: assembling the micro-nano particles on the first surface.

[0133] Next, a specific embodiment will be used to illustrate the implementation process of the method for preparing the cell electrical signal detection chip provided by the embodiments of the present invention.

[0134] First step: Form a first mask layer 102 on the substrate 101, as Figure 6A shown.

[0135] Second step: Deploy self-assembled microspheres 103 at the substrate 101 corresponding to multiple openings, as Figure 6B shown. Since the first mask layer 102 is to be removed, the self-assembled microspheres 103 on the first mask layer 102 will not affect the preparation of the microneedle structure.

[0136] Third step: Simultaneously etch the self-assembled microspheres and the substrate 101 at the substrate 101 corresponding to each opening through a dry etching process, so as to form a microneedle structure 104 for each electrode contact at the substrate 101 corresponding to each opening, and remove the first mask layer 102, as Figure 6C shown.

[0137] Fourth step: Form a second mask layer 105 on the substrate 101, as Figure 6D shown.

[0138] Fifth step: Form a conductive layer 106, as Figure 6E shown. The conductive layer 106 will cover the substrate 101, the second mask layer 105, and the microneedle structure 104. The conductive layer covering the substrate 101 will ultimately constitute the conductive structures corresponding to each pit, and the conductive layer covering the microneedle structure 104 and the covered microneedle structure 104 constitute the electrode contact 107.

[0139] Sixth step: Remove the second mask layer 105, as Figure 6F shown. After the second mask layer 105 is removed, a part of the substrate 101 will be exposed to prepare an insulating layer to isolate the conductive structures corresponding to each electrode contact.

[0140] Seventh step: Prepare an insulating layer 108, as Figure 6G shown. An insulating layer 108 is formed on the substrate 101 and the conductive layer 106.

[0141] Step 8: Prepare the limiting structure layer 109, as Figure 6H shown. The limiting structure layer 109 is formed on the insulating layer 108. The limiting structure layer 109 includes various grooves (not shown in the figure). Finally, various pits 110 can be obtained. While preparing the limiting structure layer 109, grooves connecting adjacent pits 110 will be obtained.

[0142] Figure 7 is a schematic flow chart of the cell limiting inoculation method provided in the sixth embodiment of the present invention. As Figure 7 shown, the cell limiting inoculation method provided in the embodiment of the present invention is applied to the cell electrical signal detection chip described in any of the above embodiments, and includes:

[0143] S701: Drop the cell suspension on the limiting structure layer;

[0144] Specifically, drop the cell suspension at the limiting structure. The concentration of the cell suspension is selected according to actual needs, and the embodiments of the present invention do not make any limitations.

[0145] S702: Centrifuge the cell electrical signal detection chip so that the cells in the cell suspension settle and enter the pits;

[0146] Specifically, centrifuge the cell electrical signal detection chip so that the cells in the cell suspension settle and enter the pits. By centrifugation, the cells in the cell suspension can be accelerated to enter the pits.

[0147] For example, the cell electrical signal detection chip can be placed on a centrifuge and rotated. Gradually accelerate the centrifuge speed to a preset speed so that the centrifugal force reaches a preset centrifugal force, and the preset centrifugal force is greater than 200g and less than or equal to 300g.

[0148] S703: If the centrifugation stop condition is met, stop the centrifugation of the cell electrical signal detection chip and remove the supernatant on the limiting structure layer.

[0149] Specifically, after cells enter each pit, there will still be cells outside. After the centrifugation stop condition is met, the centrifugation of the cell electrical signal detection chip can be stopped. Add an appropriate amount of culture medium to suspend the excess cells, and then aspirate the supernatant to remove the supernatant on the limiting structure layer. For the cells remaining in the pits, an appropriate amount of culture medium can be added for normal culture. Among them, the centrifugation stop condition is preset and set according to actual needs, and the embodiments of the present invention do not make any limitations.

[0150] For example, by rotating the cell electrical signal detection chip with a centrifuge, the centrifugation stop condition is: when the centrifuge speed is accelerated to the preset speed, or the centrifugal force reaches the preset centrifugal force.

[0151] The cell-limited inoculation method provided by the embodiments of the present invention speeds up the entry of cells into the pits through centrifugation, improving the cell inoculation efficiency.

[0152] On the basis of the above embodiments, further, a static cell electrical signal detection chip is used to replace the rotating cell electrical signal detection chip to prevent cell damage.

[0153] Specifically, for fragile cells, centrifuging the cell electrical signal detection chip is likely to cause cell damage. The cell electrical signal detection chip can be static, without centrifuging the cell electrical signal detection chip, allowing the cells to naturally settle into the pits under the influence of gravity.

[0154] In the description of this specification, the descriptions with reference to terms such as "one embodiment", "a specific embodiment", "some embodiments", "for example", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0155] The above specific embodiments have further elaborated on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cell electrical signal detection chip, characterized in that It includes a substrate, an insulating layer, a limiting structure layer and a plurality of pits, wherein: The substrate, the insulating layer and the limiting structure layer are stacked in sequence. The plurality of pits penetrate through the insulating layer and the limiting structure layer, and adjacent pits are connected by grooves; an electrode contact is arranged in each pit, each pit has a corresponding conductive structure, and the electrode contact arranged in each pit is electrically connected to the conductive structure corresponding to each pit; the conductive structures corresponding to the respective pits are arranged between the substrate and the insulating layer and are independent of each other; The pits are used to accommodate and limit single cells, and the grooves are used to limit synaptic connections between cells.

2. The cell electro-signal detection chip according to claim 1, wherein The plurality of pits are arranged in an array.

3. The cell electro-signal detection chip according to claim 1, wherein The pits are cylindrical.

4. The cell electro-signal detection chip according to claim 3, wherein The diameter of the pits is greater than the circumscribed circle diameter of a single cell and less than 2 times the circumscribed circle diameter of a single cell.

5. The cell electro-signal detection chip according to claim 3, wherein The width of the grooves is less than the circumscribed circle diameter of a single cell.

6. The cell electro-signal detection chip according to any one of claims 1 to 5, characterized in that The electrode contacts are needle-like structures.

7. A method for preparing a cell electrical signal detection chip, applied to the cell electrical signal detection chip according to any one of claims 1 to 6, characterized in that, It includes: Form a first mask layer on the substrate. The first mask layer includes a plurality of openings, and the openings correspond to the electrode contacts one by one; Form microstructures of a plurality of electrode contacts at the substrate corresponding to the plurality of openings and remove the first mask layer; Form a second mask layer on the substrate; Form a conductive layer and remove the second mask layer to expose the corresponding substrate; wherein, the conductive layer on the substrate constitutes a circuit layer; Form an insulating layer on the circuit layer and the substrate; Form a limiting structure layer on the insulating layer, and the limiting structure layer includes each groove.

8. The method according to claim 7, wherein Forming microstructures of a plurality of electrode contacts at the substrate corresponding to the plurality of openings and removing the first mask layer includes: Deploy self-assembled microspheres at the substrate corresponding to the plurality of openings; Perform dry etching on the self-assembled microspheres at the substrate corresponding to each opening to obtain micro-needle structures of the electrode contacts.

9. The method according to claim 7, wherein At least one of the first mask layer, the second mask layer, the insulating layer and the limiting structure layer is prepared by a micro-nano particle patterning process; the micro-nano particle patterning process includes: Assemble micro-nano particles on the first surface of the first object or the second surface of the second object to form a micro-nano particle layer; wherein, a preset transfer pattern is provided on the second surface; Stack the first object and the second object so that the micro-nano particle layer is located between the first surface and the second surface; Press the first object and the second object, wherein a part of the micro-nano particle layer corresponding to the preset transfer pattern is combined with the first object, and another part of the micro-nano particle layer is combined with the second object; Separate the first object and the second object to obtain a patterned micro-nano particle array on the first object, wherein the pattern of the micro-nano particle array is consistent with the preset transfer pattern; Modify the micro-nano particle array to obtain a mask layer tightly combined with the first object.

10. The method according to claim 9, characterized in that, The modifying the micro-nano particle array to obtain a mask layer tightly combined with the first object includes: Adopt a thermal sintering method to completely melt and re-plasticize the micro-nano particles of the micro-nano particle array to obtain a dense mask layer; The micro-nano particles of the micro-nano particle array are partially melted and re-shaped by a thermal sintering method to obtain a porous mask layer; or, The micro-nano particle array is modified by a chemical modification method to obtain a mask layer.

11. A method for limiting cell inoculation of the cell electrical signal detection chip according to any one of claims 1 to 6, characterized in that, Comprising: Dripping a cell suspension on the limiting structure layer; Centrifuging the cell electro-signal detection chip so that the cells in the cell suspension settle and enter the pits; Stopping the centrifugation of the cell electro-signal detection chip and removing the supernatant on the limiting structure layer.

12. The cell-limited inoculation method according to claim 11, wherein, Using a static cell electro-signal detection chip to replace the centrifuged cell electro-signal detection chip to prevent cell damage.

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