A biochip with nano-pore and micro-channel structure and a preparation method thereof

Abstract

The application provides a biochip with a nano-pore and a micro-channel structure and a preparation method thereof, the biochip uses a channel structure formed by nano-pores to accurately focus an electric field on a cell surface, reversibly opens a hydrophilic channel with a diameter corresponding to that of the nano-pores on the cell membrane surface, accelerates the entry of drug molecules into the cell through dielectrophoresis through the first electrode and the second electrode, and then the cell realizes the closure of the cell membrane opening through self-repair, so as to realize efficient, safe and accurate controllable delivery and transfection of macromolecules of living cells; and the micro-channel formed by the encapsulation of the cover plate and the substrate can ensure the flow and distribution of the cells in the micro-channel, and can also ensure that the cells can produce sufficient deformation and closely adhere to the nano-pores on the substrate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochip technology, and more particularly to a biochip with nano-pore and micro-channel structure and a preparation method thereof. BACKGROUND

[0002] Cells have complex microstructures and carry important genetic information. Transfection and drug delivery in cells provide effective tools for various researches such as cell therapy, genome editing and biosynthesis. Traditional large-scale drug delivery systems have various problems such as low cell survival rate, large cytotoxic side effects and poor uniformity. Precise manipulation based on cell level can effectively reduce the damage to cells, and targeted drug delivery in single cell dimension can improve efficiency and consistency.

[0003] In the nucleic acid delivery carrier system of cells, there are virus carriers, chemical carriers, exosome carriers and physical carriers, etc. The virus carrier has the characteristics of wide application range, high throughput and stable transfection integrated into the genome, and is most commonly used for cell and gene therapy. For cell and gene therapy (CGT), the virus carrier is the most fully verified technology route at present. However, the virus carrier also has many shortcomings and deficiencies, such as natural immune mechanism, off-target toxic side effects, and high cost, etc. The chemical carrier is mainly based on lipid nanoparticles (LNP), which has the characteristics of mature process and high encapsulation and release efficiency. However, its shortcomings are high process cost and that the core technology is mastered by many companies. At the same time, it can only deliver small fragment length nucleic acid drugs. The exosome carrier is a vesicle endogenously produced in human cells, which is the core of cell communication and has therapeutic potential. In recent years, it has attracted widespread attention as a gene molecular drug delivery carrier. Its advantages are that it can carry DNA, RNA and protein functional molecules, has low immunogenicity and high functional activity. Its disadvantage is that the production process is extremely complex, and it is difficult to engineer and modify the exosome.

[0004] For the physical carrier of cell delivery, the main method in the industry is based on the traditional electric shock cup. This method needs to mix the target cells to be processed, cell culture solution, special formula of electrotransformation solution and the substances to be delivered together, and then add the cell mixture to the electric shock cup. A specific high-voltage electric field is applied to the electric shock cup. The high-voltage electric field randomly processes a large number of cells in the cell mixture. Overall, the traditional electric conversion method is simple and direct, but has the shortcomings of random transfection process, low transfection efficiency and large cell damage. SUMMARY

[0005] In view of the above, in order to solve the above problems, the present application provides a biochip with nano-pores and micro-channel structure and a preparation method thereof, and the technical scheme is as follows:

[0006] A biochip with nano-pores and micro-channel structure, comprising:

[0007] a substrate with a first region and a second region to be formed into a chip, the first region having a plurality of nano-pores;

[0008] a cover plate arranged opposite to the substrate, a micro-channel being formed between the cover plate and the substrate, the micro-channel comprising a first micro-channel region and a second micro-channel region, the size of the first micro-channel region being larger than that of the second micro-channel region;

[0009] a first electrode located on the side of the nano-pores away from the micro-channel, and a second electrode located on the side of the micro-channel away from the substrate;

[0010] wherein the cells flow from the first micro-channel region to the second micro-channel region.

[0011] Preferably, in the above biochip, the plurality of nano-pores are arranged in an array.

[0012] Preferably, in the above biochip, the substrate is a silicon material substrate, and the cover plate is a glass cover plate.

[0013] Preferably, in the above biochip, the back surface of the second region has a first reinforcing rib and a second reinforcing rib;

[0014] the first reinforcing rib extends along a first direction and is arranged in sequence along a second direction;

[0015] the second reinforcing rib extends along the second direction and is arranged in sequence along the first direction;

[0016] the first direction and the second direction intersect.

[0017] Preferably, in the above biochip, the first micro-channel region comprises a first region and a second region;

[0018] in the first region, the distance between the cover plate and the substrate is equal;

[0019] in the second region, the distance between the cover plate and the substrate gradually decreases.

[0020] Preferably, in the above biochip, in the second micro-channel region, the distance between the cover plate and the substrate is equal;

[0021] In the second region, the minimum distance between the cover plate and the substrate is H1;

[0022] In the second microfluidic channel region, the distance between the cover plate and the substrate is H2;

[0023] H1 = H2.

[0024] A method for preparing a biochip with nano-pores and microfluidic channels, for preparing the biochip of any one of the above, the method comprising:

[0025] providing a substrate, the substrate having a first region and a second region to be formed into a chip;

[0026] treating the substrate to form a plurality of nano-pores on the first region;

[0027] providing a cover plate and treating the cover plate to form a microfluidic channel between the cover plate and the substrate after bonding the cover plate and the substrate, the microfluidic channel comprising a first microfluidic channel region and a second microfluidic channel region, the first microfluidic channel region having a larger size than the second microfluidic channel region;

[0028] wherein a first electrode is further prepared on a side of the nano-pores away from the microfluidic channel, and a second electrode is further prepared on a side of the microfluidic channel away from the substrate; and cells flow from the first microfluidic channel region to the second microfluidic channel region.

[0029] Preferably, in the above method, after forming the nano-pores, the method further comprises:

[0030] forming a first reinforcing rib and a second reinforcing rib on the back of the second region; the first reinforcing rib extends along a first direction and is arranged in sequence along a second direction; the second reinforcing rib extends along the second direction and is arranged in sequence along the first direction; the first direction and the second direction intersect.

[0031] Compared with the prior art, the present application has the following advantages:

[0032] The application provides a biochip with a nano-pore and a micro-channel structure, which comprises a substrate, wherein the substrate has a first region and a second region to be formed into a chip, and the first region has a plurality of nano-pores; a cover plate is arranged opposite to the substrate, and a micro-channel is formed between the cover plate and the substrate, wherein the micro-channel comprises a first micro-channel region and a second micro-channel region, and the size of the first micro-channel region is larger than that of the second micro-channel region; a first electrode is arranged on a side of the nano-pore away from the micro-channel, and a second electrode is arranged on a side of the micro-channel away from the substrate; and cells flow from the first micro-channel region to the second micro-channel region. The channel structure formed by the nano-pore can accurately focus an electric field on the surface of the cell, so that a hydrophilic channel with a diameter corresponding to that of the nano-pore is reversibly opened on the surface of the cell membrane, then the drug molecules are accelerated to enter the cell through dielectrophoresis by the first electrode and the second electrode, and then the cell membrane opening is closed by the repair of the cell, so that efficient, safe and accurate controllable delivery and transfection of macromolecules of living cells are realized. The micro-channel formed by the cover plate and the substrate can ensure the flow and distribution of the cells in the micro-channel, and can also ensure that the cells can produce sufficient deformation and tightly adhere to the nano-pores on the substrate. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of the provided drawings.

[0034] Figure 1 A top view structural schematic diagram of a biochip with a nano-pore and a micro-channel structure provided by the embodiment of the present application;

[0035] Figure 2 A cross-sectional structural schematic diagram of a biochip with a nano-pore and a micro-channel structure provided by the embodiment of the present application;

[0036] Figure 3 A schematic diagram of a contact state of a cell with a nano-pore after the cell is compressed in a second channel region provided by the embodiment of the present application;

[0037] Figure 4 A simulation calculation structural schematic diagram of an electric field generating a pressure difference on a cell membrane through a nano-pore provided by the embodiment of the present application;

[0038] Figure 5 A distribution schematic diagram of a cell in a micro-channel of the biochip with a nano-pore and a micro-channel structure under a microscope provided by the embodiment of the present application;

[0039] Figure 6 This is a schematic diagram of a nanopore arrangement provided in an embodiment of the present invention;

[0040] Figure 7 This is a schematic diagram of the physical arrangement of nanopores provided in an embodiment of the present invention;

[0041] Figure 8 A cross-sectional schematic diagram of a nanopore arrangement provided in an embodiment of the present invention;

[0042] Figure 9 A schematic diagram of a reinforcing rib on the back of a second region provided in an embodiment of the present invention;

[0043] Figure 10 A schematic diagram of another reinforcing rib on the back of the second region provided in an embodiment of the present invention;

[0044] Figure 11 A schematic flowchart illustrating a method for fabricating a biochip with nanopores and microfluidic structures according to an embodiment of the present invention;

[0045] Figure 12 A method provided by an embodiment of the present invention Figure 11 The diagram shows a simplified process step corresponding to the preparation method shown. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0048] refer to Figure 1 , Figure 1 This is a top view schematic diagram of a biochip with nanopores and microfluidic channels provided in an embodiment of the present invention, with reference to... Figure 2 , Figure 2 A schematic cross-sectional view of a biochip with nanopores and microfluidic channels provided in this embodiment of the invention; see reference. Figure 3 , Figure 3 This is a schematic diagram illustrating the contact state between a cell and a nanopore after compression within the second flow channel region, as provided in an embodiment of the present invention; see reference. Figure 4 , Figure 4This is a schematic diagram of a simulation calculation structure for an electric field generating a pressure difference in a cell membrane through a nanopore, provided in an embodiment of the present invention; Reference Figure 5 , Figure 5 This is a schematic diagram of cell distribution under a microscope inside the microchannels of a biochip with nanopores and microchannel structures, provided as an embodiment of the present invention.

[0049] This biochip with nanopores and microfluidic structures includes:

[0050] like Figure 1 As shown, substrate 1 has a first region 3 and a second region 2 to be formed into a chip. The first region 3 has a plurality of nanopores 10, that is, the first region 3 is used to form nanopores 10.

[0051] like Figure 2 As shown, a cover plate 14 is disposed opposite to the substrate 13, and a microchannel is formed between the cover plate 14 and the substrate 13. The microchannel includes a first microchannel region 15 and a second microchannel region 16. The size of the first microchannel region 15 is larger than the size of the second microchannel region 16. It should be noted that... Figure 2 The reference numeral 13 indicates a substrate 1 with nanopores.

[0052] A first electrode located on the side of the nanopore 10 away from the microchannel, and a second electrode located on the side of the microchannel away from the substrate 1.

[0053] In this process, the cells flow in a direction 20 from the first microfluidic channel region 15 to the second microfluidic channel region 16. It should be noted that label 17 indicates the cell state before compression; label 18 indicates the cell state after compression.

[0054] Optional, such as Figure 2 As shown, the first microchannel region includes a first region and a second region.

[0055] In the first region, the distance between the cover plate 14 and the base 13 is equal.

[0056] In the second region, the distance between the cover plate 14 and the base 13 gradually decreases.

[0057] In the second microchannel region, the distance between the cover plate 14 and the substrate 13 is equal.

[0058] In the second region, the minimum distance between the cover plate 14 and the base 13 is H1.

[0059] In the second microchannel region, the distance between the cover plate 14 and the substrate 13 is H2.

[0060] Where H1 = H2.

[0061] Specifically, in the embodiments of the present invention, the biochip with nanopores 10 and microchannels has both nanopores 10 and microchannels, meaning that the biochip with nanopores and microchannels has two core functions: cell capture and manipulation based on the microchannel structure, and cell transfection based on the nanopores 10.

[0062] This biochip, featuring nanopores and microchannels, can be packaged and integrated to create a biochip for nanoelectroporation technology. This includes, but is not limited to, forming multiple nanopores 10 penetrating a substrate 13 on a specific material substrate using semiconductor and micro / nano fabrication processes. The channel structure formed by the nanopores 10 can precisely focus an electric field onto the cell surface, reversibly opening hydrophilic channels on the cell membrane surface with a diameter equivalent to the nanopores 10. Drug molecules are then accelerated into the cell via dielectrophoresis through the first and second electrodes. The cell then closes the cell membrane openings through its own repair process, achieving efficient, safe, precise, and controllable delivery and transfection of macromolecules in live cells. Furthermore, the microchannels formed by the cover plate 14 and the substrate 13 ensure cell flow and distribution within the microchannels, while also ensuring sufficient deformation of the cells to tightly adhere to the nanopores 10 on the substrate 13. Figure 3 As shown in number 19, this represents the contact state between the compressed cell and the nanopore.

[0063] It should be noted that, as Figure 4 As shown, taking the direction of the electric field 21 from the first electrode to the second electrode as an example, the channel structure formed by the nanopores 10 can precisely focus the electric field on the cell surface, allowing the cell membrane surface to reversibly open hydrophilic channels with a diameter equivalent to that of the nanopores (such as...). Figure 4 The effect shown in reference numeral 22 (i.e., the effect of voltage acting on the cell membrane) is then accelerated by the first and second electrodes, allowing drug molecules to enter the cell via dielectrophoresis; for example... Figure 5 As shown, label 23 indicates the distribution pattern of cells under a microscope.

[0064] Optionally, in another embodiment of the invention, reference is made to... Figure 6 , Figure 6 This is a schematic diagram of a nanopore arrangement provided in an embodiment of the present invention, with reference to... Figure 7 , Figure 7 This is a schematic diagram of the physical arrangement of nanopores provided in an embodiment of the present invention; see reference. Figure 8 , Figure 8 This is a cross-sectional schematic diagram of a nanopore arrangement provided in an embodiment of the present invention.

[0065] Multiple nanopores are arranged in a 10-point lattice.

[0066] Specifically, in this embodiment of the invention, multiple nanopores 10 arranged in a lattice can uniformly apply an electric field to the cell membrane, such as... Figure 8 As shown, reference numeral 11 indicates the sidewall of nanopore 10, and reference numeral 12 indicates the bottom of nanopore.

[0067] Optionally, in another embodiment of the present invention, the substrate 13 includes, but is not limited to, a silicon material substrate; and the cover plate 14 includes, but is not limited to, a glass cover plate.

[0068] It should be noted that, in this embodiment of the invention, the substrate 13 is described as a silicon material substrate, and the cover plate 14 is described as a glass cover plate.

[0069] Optionally, in another embodiment of the invention, reference is made to... Figure 9 , Figure 9 This is a schematic diagram of a reinforcing rib on the back of a second region provided in an embodiment of the present invention, with reference to... Figure 10 , Figure 10 This is a schematic diagram of another reinforcing rib on the back of the second region provided in an embodiment of the present invention.

[0070] The back of the second region 2 has a first reinforcing rib and a second reinforcing rib, such as Figure 9 The first reinforcing rib 5 and the second reinforcing rib 6 shown, or as... Figure 10 The first reinforcing rib 8 and the second reinforcing rib 9 are shown in the figure.

[0071] The first reinforcing rib extends along the first direction and is arranged sequentially along the second direction.

[0072] The second reinforcing rib extends along the second direction and is arranged sequentially along the first direction.

[0073] The first direction and the second direction intersect.

[0074] Specifically, in this embodiment of the invention, taking the first direction being perpendicular to the second direction as an example, the first reinforcing rib and the second reinforcing rib form multiple back cavity grooves (e.g., Figure 9 The back cavity groove 4 shown, or as Figure 10 The back cavity groove 7 shown in the figure can be sized according to the actual situation, such as Figure 10 The dimensions of the back cavity groove 7 shown are larger than those shown. Figure 9 The dimensions of the back cavity groove 4 shown indicate that by employing front-side nanopore photolithography and back-side back cavity groove reinforcing ribs, it is possible to ensure the formation of high aspect ratio nanopores on the substrate and to ensure the strength of the area where the nanopores are located on the substrate, thereby improving the structural stability of the substrate.

[0075] Optionally, based on the above embodiments of the present invention, another embodiment of the present invention also provides a method for fabricating a biochip with nanopores and microfluidic structures, used to fabricate the biochip described in the above embodiments, referencing... Figure 11 , Figure 11 This is a schematic flowchart illustrating a method for fabricating a biochip with nanopores and microfluidic structures according to an embodiment of the present invention. The fabrication method includes:

[0076] S101: Provide a substrate having a first region and a second region to be formed into a chip.

[0077] S102: The substrate is processed to form multiple nanopores in the first region.

[0078] S103: A cover plate is provided and processed to form a microchannel between the cover plate and the substrate after the cover plate and the substrate are bonded. The microchannel includes a first microchannel region and a second microchannel region. The size of the first microchannel region is larger than the size of the second microchannel region. A first electrode is also prepared on the side of the nanopore away from the microchannel, and a second electrode is also prepared on the side of the microchannel away from the substrate. Cells flow from the first microchannel region to the second microchannel region.

[0079] Optionally, in another embodiment of the present invention, after forming the nanopores, the preparation method further includes:

[0080] A first reinforcing rib and a second reinforcing rib are formed on the back side of the second region; the first reinforcing rib extends along a first direction and is arranged sequentially along a second direction; the second reinforcing rib extends along the second direction and is arranged sequentially along the first direction; the first direction and the second direction intersect.

[0081] For details, please refer to Figure 12 , Figure 12 A method provided by an embodiment of the present invention Figure 11 The diagram illustrates a simplified process step corresponding to the fabrication method shown. First, a substrate, which can be a silicon wafer, is provided. Then, nanopore photolithography is performed based on a nanopore mask, followed by nanopore etching. At this point, nanopores with a certain depth are formed on the front side of the substrate, but the nanopores do not penetrate the substrate. Next, back cavity photolithography and back cavity etching are performed on the back side of the substrate based on a back cavity mask until the nanopores are exposed. At this point, the nanopores completely penetrate the substrate, forming the required lattice arrangement of nanopores. Optionally, a first reinforcing rib and a second reinforcing rib can also be formed on the back side of a second region of the substrate using semiconductor processes. This completes the substrate fabrication process.

[0082] While processing the substrate, the cover plate can also be processed simultaneously. The cover plate can be a glass wafer. The morphology of one side of the cover plate is processed based on the microchannel mask, that is, microchannel photolithography and microchannel etching, as well as semiconductor processes such as inlet and outlet processing and electrode evaporation.

[0083] After the cover plate and substrate are processed, wafer alignment and bonding processes are performed, followed by chip dicing to form the desired biochip with nanopores and microfluidic structures.

[0084] As described above, the technical solution of the biochip with nanopores and microfluidic channels and its preparation method provided by the embodiments of the present invention includes a design scheme for the biochip and a processing and preparation method for the nanopore and microfluidic channels. The biochip with nanopores and microfluidic channels can effectively control cell movement and perform corresponding transfection and editing of cells. In terms of processing and preparation, the biochip with nanopores and microfluidic channels integrates semiconductor processing technology, microfluidic cell manipulation methods, and compatibility with biochemical reagents, which can ensure low-cost mass production, precise cell control, and efficient cell transfection processing.

[0085] It should be noted that the following points should be considered in the design and fabrication of silicon-based (i.e., silicon material substrate) nanopores in the embodiments of this invention:

[0086] The electrical conductivity, pore size, interpore spacing, and height of silicon-based materials all affect their electrical resistance, indirectly influencing the electric field distribution and their effect on cells. The nanopores prepared in this invention comprehensively consider these factors. Based on theoretical simulations and considering cell size and morphology, an optimal design was derived. In terms of fabrication, a reinforcing rib design with a back cavity was introduced, effectively improving the porosity of all nanopores and significantly enhancing the strength and yield of this silicon-based nanopore structure, thus ensuring the integration of silicon-based nanopores into biochips.

[0087] It should be noted that the following points should be considered in the design and fabrication of the microchannel structure in the embodiments of the present invention:

[0088] The main purpose of microfluidic structures is to control cell movement and distribution. Cell size is non-uniform and normally distributed, and cell movement is irregular and randomly distributed. In order to precisely manipulate cells, the size of the microfluidic structure needs to be designed to be comparable to or smaller than the cell size. The biochip provided in this invention can be designed with corresponding specifications according to different cell types to ensure the corresponding distribution and size compression of cells. The realization of this high-precision microfluidic structure requires special processing procedures and preparation methods. The microfluidic structure processing scheme proposed in this invention can precisely control its size and parameters, and can realize the gradual compression deformation of cells by the inclined structure. On the one hand, it can ensure effective deformation of cells so that the cells and silicon-based nanopores fit tightly together. On the other hand, it can prevent the channel from being blocked, thereby improving the cell processing efficiency.

[0089] In summary, this biochip with nanopores and microchannels utilizes the channel structure formed by nanopores to precisely focus an electric field on the cell surface, causing the cell membrane surface to reversibly open hydrophilic channels with a diameter equivalent to that of the nanopores. Then, through the first and second electrodes, drug molecules are accelerated to enter the cell via dielectrophoresis. Subsequently, the cell repairs itself to close the openings in the cell membrane, thereby achieving efficient, safe, precise and controllable delivery and transfection of macromolecules in live cells. Furthermore, the microchannels formed by the cover plate and substrate encapsulation ensure the flow and distribution of cells within the microchannels, while also ensuring that the cells can generate sufficient deformation and adhere tightly to the nanopores on the substrate.

[0090] Furthermore, its processing method utilizes semiconductor technology and processes, including photomask lithography, deep hole etching, and electrode evaporation, which can achieve mass production and relatively high yield. Its packaging method adopts microfluidic technology and principles, including structural design, flow channel processing, and chip packaging, while incorporating the characteristics of biological cells, including cell manipulation, electric field control, and reagent compatibility.

[0091] The present invention provides a detailed description of a biochip with nanopores and microfluidic channels and its preparation method. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

[0092] It should be noted that the various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0093] It should also be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that elements inherent to a process, method, article, or apparatus that comprises a list of elements, or elements inherent to such processes, methods, articles, or apparatus, are also included. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0094] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A biochip having a nano-pore and a micro-fluidic structure, characterized by, The biochip comprises: a substrate having a first region and a second region to be formed into a chip, the first region having a plurality of nanopores for focusing an electric field on a cell surface to reversibly open a hydrophilic channel of a cell membrane with a diameter comparable to that of the nanopores, the nanopores being formed by front nanopore lithography, back cavity lithography and back cavity etching; a back surface of the second region having a first reinforcing rib and a second reinforcing rib; the first reinforcing rib extending along a first direction and arranged in sequence along a second direction; the second reinforcing rib extending along the second direction and arranged in sequence along the first direction; the first direction and the second direction intersecting; a cover plate arranged opposite to the substrate, a microchannel being formed between the cover plate and the substrate, the microchannel including a first microchannel region and a second microchannel region, the first microchannel region having a size larger than that of the second microchannel region; a first electrode located on a side of the nanopores away from the microchannel and a second electrode located on a side of the microchannel away from the substrate, the first electrode and the second electrode being used to accelerate the entry of drug molecules into cells by dielectrophoresis; wherein the cells flow from the first microchannel region to the second microchannel region; a plurality of the nanopores arranged in an array; the first microchannel region including a first region and a second region; in the first region, the distance between the cover plate and the substrate being equal; in the second region, the distance between the cover plate and the substrate gradually decreasing; in the second microchannel region, the distance between the cover plate and the substrate being equal; in the second region, the minimum distance between the cover plate and the substrate being H1; in the second microchannel region, the distance between the cover plate and the substrate being H2; wherein H1=H2.

2. The biochip according to claim 1, wherein The substrate is a silicon material substrate; and the cover plate is a glass cover plate.

3. A method for producing a biochip having a nano-pore and a micro-fluidic structure, characterized by, A method for preparing the biochip of claim 1 or 2, the method comprising: providing a substrate having a first region and a second region to be formed into a chip; treating the substrate to form a plurality of nanopores on the first region, the nanopores being used to focus an electric field on a cell surface to reversibly open a hydrophilic channel of a cell membrane with a diameter comparable to that of the nanopores; forming a first reinforcing rib and a second reinforcing rib on a back surface of the second region; the first reinforcing rib extending along a first direction and arranged in sequence along a second direction; the second reinforcing rib extending along the second direction and arranged in sequence along the first direction; the first direction and the second direction intersecting; providing a cover plate and treating the cover plate to form a microchannel between the cover plate and the substrate after bonding the cover plate and the substrate, the microchannel including a first microchannel region and a second microchannel region, the first microchannel region having a size larger than that of the second microchannel region; Wherein, the first electrode is prepared on the side of the nanopore far from the microchannel, and the second electrode is prepared on the side of the microchannel far from the substrate; the cell flows from the first microchannel region to the second microchannel region.

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