Film-forming microstructure for nanopore gene sequencing

Abstract

The utility model provides a film-forming microstructure for nanopore gene sequencing, which comprises micro-support structure units arranged on a substrate, and each micro-support structure unit comprises a support cavity structure arranged on an upper material layer and a micro-well structure arranged on a lower material layer and corresponding to the support cavity structure, the supporting cavity structure comprises a pore channel structure; the micro-well structure comprises a film forming cavity and a fluid storage cavity, the film forming cavity and the pore channel structure are correspondingly arranged in a matched mode, the fluid storage cavity communicates with the film forming cavity, the fluid storage cavity penetrates through the lower material layer, and at least one communicating channel is formed between the film forming cavity and the fluid storage cavity; therefore, the film-forming microstructure provided by the utility model has the technical effects that the film-forming stability can be ensured, and the volume of the fluid stored in the micro-well structure can be ensured to be large enough as much as possible, so that the sequencing time of a single nanopore is prolonged, and the output efficiency of nanopore sequencing is greatly improved.

Description

Technical Field

[0001] The present application relates to the technical field of gene sequencing, and in particular to a film-forming microstructure for nanopore gene sequencing. Background Art

[0002] Gene sequencing technology uses nanopores as biosensors and embeds them into insulating amphiphilic molecular membranes. When preparing the molecular membrane of the gene sequencing chip, polar solvents, non-polar solvents of amphiphilic materials and polar solvents are usually introduced into the film-forming area of ​​the gene sequencing chip in sequence, so that the non-polar solvent of the amphiphilic material is sandwiched between two layers of polar solvent to form a molecular membrane. When a single-stranded DNA molecule passes through the nanopore, the different current changes caused by different base groups are read by the signal receiver in the nanopore, and then the corresponding base recognition is performed to achieve the detection of gene sequences. It has the advantages of high throughput, ultra-long read length, direct detection of base methylation modification, and small size for easy carrying. The formation of a stable and uniformly thick amphiphilic molecular membrane can provide a stable platform for the embedding of nanopores and single-molecule perforation, which is one of the necessary conditions to ensure the accuracy of gene detection, and the micro-support structure of the molecular membrane plays a decisive role in the stability of the molecular membrane.

[0003] The micro-support structure of the molecular membrane needs to balance between the film-forming properties and the volume of the fluid in the downwardly recessed micro-well structure. For example, the nanopore support structure disclosed in US2015 / 265994 is composed of an internal part and an external part, wherein the internal part defines an internal recess without gaps during the period, and the internal part can limit the volume of polar media that can be arranged in adjacent internal recesses to contact each other; the external part extends outward from the internal part and has the gap that allows non-polar media to flow between the compartments through the gap. In this nanopore support structure, the amphiphilic molecular membrane extends across the opening of the internal recess, and the actual film-forming size is large, which makes the film-forming capacitance large and the molecular membrane structure unstable. If the size of the inner recess is reduced, it will result in a small amount of fluid storage in the inner recess, a small sequencing reaction system, which is not conducive to the continuous sequencing, and thus greatly limits the design of the micro-support structure.

[0004] Based on this, researchers in this field urgently need to develop a new film-forming microstructure for nanopore gene sequencing, which can ensure the stability of the film while ensuring that the volume of the fluid stored in the microwell structure is large enough to extend the sequencing time of a single nanopore and improve the output efficiency of nanopore sequencing. Summary of the invention

[0005] In view of this, in order to solve the above-mentioned technical problems, the embodiments of the present application provide a film-forming microstructure for nanopore gene sequencing, which ensures the stability of the film while ensuring that the volume of the fluid stored in the microwell structure is large enough to extend the sequencing time of a single nanopore and improve the output efficiency of nanopore sequencing, thereby solving the limitation problem of micro-support structure design in the prior art.

[0006] To achieve the above-mentioned objectives, on the one hand, an embodiment of the present application provides a film-forming microstructure for nanopore gene sequencing, including a micro-support structure unit arranged on a substrate, wherein the micro-support structure unit includes a supporting cavity structure arranged on an upper material layer and a micro-well structure corresponding to the supporting cavity structure arranged on a lower material layer, wherein the supporting cavity structure includes a channel structure; the micro-well structure includes a film-forming cavity arranged in correspondence with the channel structure, and a fluid storage cavity connected to the film-forming cavity, wherein the fluid storage cavity passes through the lower material layer, and at least one connecting channel is provided between the film-forming cavity and the fluid storage cavity.

[0007] Furthermore, the cross-sectional shape of the fluid storage chamber includes one or more of a circular shape, an arc shape, an annular shape or a polygonal shape.

[0008] Furthermore, the fluid in the fluid storage chamber is in direct contact with the detection electrode of the substrate, thereby realizing the transmission and collection of sequencing electrical signals.

[0009] Furthermore, the film-forming cavity passes through or does not pass through the lower material layer, but does not directly contact the detection electrode of the substrate.

[0010] Furthermore, a plurality of exhaust holes are provided on the upper material layer above the fluid storage cavity to assist in exhausting the gas in the fluid storage cavity.

[0011] Furthermore, the cross-sectional shape of the connecting channel is a straight shape with a uniform opening from the fluid storage chamber to the film-forming chamber, and / or a gradually increasing duckbill shape, and / or a dumbbell shape with a small middle and large ends.

[0012] Furthermore, the width of the pore structure is D1, the width of the film-forming cavity is D2, and the width of the fluid storage cavity is D3, and the relationship between them is D1 <D2<D3。

[0013] Furthermore, the ratio of the width D1 of the pore structure to the width D2 of the film-forming cavity is between 1:1.5 and 1:10.

[0014] Furthermore, the ratio between the width D2 and the depth H2 of the film-forming cavity satisfies D2 / H2≥1:3, and the depth H2 of the film-forming cavity ranges from 50 μm to 1000 μm.

[0015] Furthermore, the ratio between the width D3 and the depth H3 of the fluid storage chamber is in the range of 1:1 to 1:10.

[0016] Furthermore, the width w of the connecting channel ranges from 1 to 100 μm; the depth of the connecting channel is between the depth of the film-forming cavity and the depth of the fluid storage cavity.

[0017] Another aspect of the present application provides a method for preparing the film-forming microstructure for nanopore gene sequencing described in the above embodiment, the preparation method comprising:

[0018] providing a substrate;

[0019] A micro-well structure including a fluid storage cavity, a film-forming cavity and a connecting channel penetrating the lower material layer is prepared on the substrate by exposure and development;

[0020] A channel structure penetrating the upper material layer is prepared by exposure and development on the lower material layer having the micro-well structure.

[0021] Compared with the prior art, the present application has the following advantages:

[0022] A new type of film-forming microstructure for nanopore gene sequencing provided by the present application has the technical effect of being able to ensure the stability of the film while ensuring that the volume of the fluid stored in the microwell structure is large enough to extend the sequencing time of a single nanopore, thereby greatly improving the output efficiency of nanopore sequencing.

[0023] Specifically, the present application provides a fluid storage cavity on the lower material layer that is connected to the film-forming cavity and penetrates the lower material layer. The fluid storage cavity can be designed to be large in size to accommodate a larger sequencing reaction system and increase the continuity of single nanopore sequencing. The film-forming cavity can be designed to be small in size to improve the stability of the molecular membrane, thereby achieving the technical effect of being able to ensure the stability of the film formation while ensuring that the volume of the fluid stored in the microwell structure is large enough to extend the sequencing time of a single nanopore, thereby greatly improving the output efficiency of nanopore sequencing.

[0024] Furthermore, by setting up the fluid storage cavity, the size of the film-forming cavity and the pore structure can be designed to be smaller, so the actual film-forming area of ​​the amphiphilic molecular membrane will be smaller and its stability will be further improved. In addition, since the design size of the film-forming cavity does not need to take into account the liquid storage volume in the cavity, a smaller micro-well structure can be selected, which plays a better supporting role for the pore structure axially suspended above the film-forming cavity, making it less likely for the upper structure to collapse downward, and increasing the rigidity and stability of the molecular membrane film-forming structure.

[0025] Furthermore, since the fluid storage chamber and the film-forming chamber are relatively independent, there is greater flexibility in the design of their respective dimensions, and a smaller film-forming chamber can be achieved, thereby achieving better fixation of the molecular film, reducing the actual film-forming area, and increasing the stability of the film; to a certain extent, it solves the contradiction between the small-size micro-support structure required for the stability of molecular film formation and the large volume required by the sequencing reaction system, gets rid of the size constraints between the two, and increases the flexibility of design. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings are used to provide a further understanding of the present application, constitute a part of the present application, and are only intended to provide an illustrative explanation and description of the present invention, and are not intended to limit the scope of the present invention. In the drawings:

[0027] Figure 1 This is a schematic diagram of the explosion structure of the film-forming microstructure used for nanopore gene sequencing in an embodiment of the present application;

[0028] Figure 2 This is a schematic cross-sectional view of a film-forming microstructure for nanopore gene sequencing in an embodiment of the present application;

[0029] Figure 3 This is a schematic diagram of the top view of the structure of the lower material layer in the embodiment of the present application;

[0030] Figure 4 For along Figure 3 Schematic diagram of the cross-sectional structure along the middle line AA;

[0031] Figure 5 This is a schematic diagram of a top view of the structure of a lower material layer in another embodiment of the present application;

[0032] Figure 6 This is a schematic diagram of a top view of the structure of a lower material layer in another embodiment of the present application;

[0033] Figure 7 This is a schematic diagram of a top view of the structure of a lower material layer in another embodiment of the present application;

[0034] Figure 8 This is a schematic diagram of a top view of the structure of a lower material layer in another embodiment of the present application;

[0035] Fig. 9 This is a schematic diagram of a top view of the structure of a lower material layer in another embodiment of the present application;

[0036] Fig.10 This is a schematic diagram of a top view of the structure of a lower material layer in another embodiment of the present application;

[0037] Fig.11 This is a schematic diagram of a top view of the structure of a lower material layer in another embodiment of the present application;

[0038] Fig.12 This is a schematic diagram of the structure of a film-forming microstructure for nanopore gene sequencing in another embodiment of the present application;

[0039] Fig.13 This is a schematic diagram of the structure of a film-forming microstructure for nanopore gene sequencing in another embodiment of the present application.

[0040] Reference numerals:

[0041] 1. Upper material layer; 10. Support cavity structure; 11. Channel structure; 12. Exhaust hole; 110. Center hole; 111. Edge hole; 112. Limiting protrusion; 113. Groove structure; 114. Neck shrinkage; 12. Fluid buffer structure; 121. Buffer tank; 123. Communication channel;

[0042] 2. Lower material layer; 20. Microwell structure; 21. Fluid storage chamber; 22. Film forming chamber; 23. Communication channel;

[0043] 3. Substrate; 30. Detection electrode. DETAILED DESCRIPTION

[0044] Several implementation methods of the present application will be disclosed below, and the technical solution of the present application will be described clearly and completely. The drawings that constitute a part of the specification of the present application are used to provide a further understanding of the present application. The schematic embodiments and descriptions of the present application are used to explain the utility model and do not constitute an improper limitation on the application. Based on the embodiments in the utility model, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present application.

[0045] It should be noted that unless a direction is defined separately, the directions such as up, down, left, and right mentioned in this document are based on the embodiments of this application. Figure 1 The directions of up, down, left, right, etc. shown are used as the basis. If the specific posture changes, the directional indication will also change accordingly. The words "first", "second", "third", "fourth" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish different components. In addition, in various embodiments of the present disclosure, the same or similar reference numerals represent the same or similar components.

[0046] In this application, unless otherwise specified and limited, the terms "connection", "fixation" and the like should be understood in a broad sense. For example, "connection" can be a fixed connection, a detachable connection, or an integral body, unless otherwise specified. For ordinary technicians in this field, the specific meanings of the above terms in this utility model can be understood according to specific circumstances.

[0047] In addition, the technical solutions between the various embodiments of the present application can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present utility model.

[0048] Embodiment 1

[0049] See also Figure 1-Figure 4 As shown, this embodiment provides a film-forming microstructure for nanopore gene sequencing, including a micro-support structure unit arranged on a substrate 3, each of the micro-support structure units including a supporting cavity structure arranged on an upper material layer 1 and a micro-well structure 20 corresponding to the supporting cavity structure arranged on a lower material layer 2, wherein the supporting cavity structure includes a pore structure 11; the micro-well structure 20 includes a film-forming cavity 22 arranged in a corresponding manner to the pore structure 11, and a fluid storage cavity 21 connected to the film-forming cavity 22, wherein the fluid storage cavity 21 passes through the lower material layer 2, and at least one connecting channel 23 is provided between the film-forming cavity 22 and the fluid storage cavity 21.

[0050] It should be noted that the cross-sectional shape of the fluid storage chamber 21 in this embodiment is a circle as an example. Figure 3 , Figure 5 , Figure 6 As shown; of course, in actual application, it can also be designed as follows Figure 7 The shuttle shape shown, such as Fig.10 The arc shown, such as Fig.11 The ring shown or Fig. 9 The polygon shown.

[0051] like Figure 3 As shown, the cross-sectional shape of the connecting channel 23 in this embodiment is a straight channel shape with a uniform opening from the fluid storage chamber 21 to the film forming chamber 22 as an example; of course, in actual application, it can also be designed as follows Figure 5 The duckbill shape gradually increases from the fluid storage chamber 21 to the film forming chamber 22, and / or is as shown in Figure 6 The stepped shape shown, and / or Figure 8 The shape shown is a dumbbell with a small middle and large ends.

[0052] Therefore, this embodiment enhances the delivery of the polar medium between the two chambers by changing the shapes of the film-forming chamber 22 and the fluid storage chamber 21 or the structural design of the connecting channel 23 for communicating the two chambers. Fig.10 , Fig.11 The number of connecting channels 23 is increased to improve the efficiency of polar medium delivery between chambers, so as to fully meet the continuous stability of the gene sequencing reaction system.

[0053] like Figure 3 As shown, the fluid storage chamber 21 in this embodiment passes through the lower material layer 2 and is connected to the substrate 3. The fluid in the fluid storage chamber can directly contact the detection electrode 30 on the substrate 3 to realize the transmission and collection of sequencing electrical signals. The fluid storage chamber can be designed in a large size to accommodate a larger sequencing reaction system and increase the continuity of single nanopore sequencing. The film-forming chamber can be designed in a small size to improve the stability of the molecular film, thereby achieving the technical effect of being able to ensure the stability of the film formation while ensuring that the volume of the fluid stored in the microwell structure is large enough to extend the sequencing time of a single nanopore, thereby greatly improving the output efficiency of nanopore sequencing.

[0054] Combination Figure 2 As shown, the channel structure 11 in this embodiment is suspended above the film-forming cavity 22, and the geometric center of the channel structure 11 is axially aligned with the geometric center of the film-forming cavity 22, and the radial dimension of the channel structure 11 is smaller than the radial dimension of the film-forming cavity 22. Since the design size of the film-forming cavity 22 does not need to take into account the liquid storage volume in the cavity, a film-forming cavity with a smaller radial dimension that penetrates or does not penetrate the lower material layer 2 can be selected, but neither is in direct contact with the detection electrode on the substrate; considering the need for wiring of wires connected to the detection electrode, it is preferred that the bottom end of the film-forming cavity is closed, that is, it does not penetrate the lower material layer; the main function of the film-forming cavity is to cooperate with the channel structure of the upper material layer 1 to promote the formation of amphiphilic molecular film, and it plays a better supporting role for the channel structure 11 axially suspended above the film-forming cavity 22, so that the upper structure is not prone to collapse downward, thereby increasing the rigidity and stability of the molecular film forming structure.

[0055] The principle of the film-forming process of the amphiphilic molecular film is briefly introduced as follows using the technical solution of the film-forming microstructure for nanopore gene sequencing provided in this embodiment:

[0056] First, the arrayed micro-support structure units are placed in a vacuum environment to expel the air in the cavity, and then a polar medium is introduced. The polar medium enters the film-forming cavity and the fluid storage cavity through the pore structure of the upper material layer. After the polar medium fills the film-forming cavity and the fluid storage cavity, the surface of the fluid in the film-forming cavity is affected by the surface tension to form a meniscus-shaped interface that bulges upward, and the meniscus-shaped interface enters the pore structure of the upper material layer.

[0057] Then, a non-polar medium is introduced, and after entering the pore structure of the upper material layer through the flow channel, a downward convex meniscus interface is formed. The two interfaces are fully connected, providing good conditions for the spreading and film formation of amphiphilic molecules.

[0058] Finally, after the polar medium is introduced, it enters the pore structure and covers the non-polar medium, thereby forming an amphiphilic molecular membrane with uniform thickness, moderate stability in the pore structure.

[0059] Embodiment 2

[0060] Based on Example 1, as a preferred implementation, Fig.12 As shown, a plurality of exhaust holes 12 are provided on the upper material layer 1 above the fluid storage cavity 21 in this embodiment to assist in the discharge of gas in the fluid storage cavity and improve the efficiency of polar media entering the fluid storage cavity.

[0061] As a preferred embodiment, Fig.13 As shown, the support cavity structure 10 in this embodiment includes a channel structure 11 and a plurality of fluid buffer structures 12 arranged at the peripheral edge of the channel structure 11 and connected to the channel structure 11, the channel structure 11 passes through the upper material layer 1, and the fluid buffer structure 12 is arranged on the upper surface of the upper material layer 1 but does not pass through the upper material layer 1. Furthermore, the fluid buffer structure 12 in this embodiment includes a buffer pool 121 and a communication channel 123 for connecting the buffer pool 121 and the channel structure 11; further, the channel structure 11 includes a central hole 110 and an edge hole 111, and the edge hole 111 is provided with a plurality of limiting protrusions 112 extending toward the central hole 110 on the hole wall close to the fluid buffer structure 12, and a groove structure 113 is formed between each adjacent two limiting protrusions 112. The setting of the limiting protrusions, the groove structure and the necking part can provide a strong supporting effect for the molecular membrane during the process of forming the molecular membrane in the supporting cavity structure, thereby greatly enhancing the stability of the combination of the membrane and the supporting cavity structure, and is beneficial to improving the film forming rate and stability of the molecular membrane after film formation.

[0062] It should be noted that, in this embodiment, a fluid buffer structure 12 connected to the pore structure 11 is arranged on the peripheral edge of the pore structure 11 of each supporting cavity structure 10 on the upper material layer 1, and the fluid buffer structure 12 is arranged on the upper surface of the upper material layer 1 but does not penetrate the upper material layer 1, thereby achieving the retention of the non-polar medium and the real-time circulation with the non-polar medium in the pore structure, achieving the flow regulation of the non-polar medium flowing into the pore structure, ensuring the thickness uniformity of the non-polar medium flowing into the pore structure, and further ensuring that there is almost no or very little difference in film formation between the micro-support structures in the array structure, thereby greatly improving the uniformity and stability of molecular film formation in the array structure.

[0063] Furthermore, the film-forming position of the molecular film is located in the pore structure 11 communicating with the fluid buffer structure 12, so that the sizes of the pore structure 11 of the upper material layer and the micro-well structure 20 of the lower material layer are no longer restricted by the film-forming area. While ensuring the film-forming stability, the volume of the micro-well structure can be increased, thereby expanding the reaction system in the micro-well structure 20, extending the nanopore sequencing time, greatly improving the flexibility of the micro-support structure design, and further enabling the molecular film-forming device of the present application to have the technical effects of overall film-forming uniformity, stability, and relatively small film-forming differences among the micro-support structures.

[0064] In addition, the groove structure 113 in this embodiment is directly connected to the buffer pool 121 through the communication flow channel 123, which is conducive to ensuring the full cooperation of the convex interface on the upper surface of the film-forming cavity appearing in the pore structure, and provides more favorable conditions for the spreading of the amphiphilic molecular layer.

[0065] Embodiment III

[0066] Based on Embodiment I and Embodiment II, as a preferred implementation manner, in combination with Figure 2 As shown, in this embodiment, the width of the pore structure 11 is D1, the width of the film-forming cavity 22 is D2, and the width of the fluid storage cavity 21 is D3. The relationship among them is D1 < D2 < D3. Further, the ratio between the width D1 of the pore structure and the width D2 of the film-forming cavity is between 1:1.5 and 1:10; the ratio relationship between the width D2 of the film-forming cavity and the depth H2 satisfies D2 / H2 ≥ 1:3, and the value range of the depth H2 of the film-forming cavity is 50 μm to 1000 μm; the ratio range between the width D3 and the depth H3 of the fluid storage cavity is between 1:1 and 1:10; the value range of the width w of the communication channel is 1 to 100 μm; the depth of the communication channel is between the depth of the film-forming cavity and the depth of the fluid storage cavity.

[0067] It should be noted that through the unique design of the fluid buffer structure and the fluid storage cavity in the present application, the sizes of the pore structure 11 of the upper material layer and the micro-well structure 20 of the lower material layer are no longer restricted by the film-forming area. While ensuring the film-forming stability, the detection requirements of reaction and extended nanopore sequencing time can be met. At the same time, combined with the film-forming cavity size designed in cooperation with the fluid storage cavity structure, it is conducive to ensuring that the ratio between the width D1 of the pore structure and the width D2 of the film-forming cavity is between 1:1.5 and 1:10 and the ratio relationship between the width D2 of the film-forming cavity and the depth H2 satisfies D2 / H2 ≥ 1:3. Furthermore, it ensures the technical effect of being able to ensure the film-forming stability while also ensuring that the volume of the fluid stored in the micro-well structure is large enough to extend the sequencing time of a single nanopore and greatly improve the output efficiency of nanopore sequencing.

[0068] Embodiment 5

[0069] An embodiment of the present application provides a method for preparing a film-forming microstructure for nanopore gene sequencing as described in the above embodiment, and the preparation method comprises: providing a substrate; preparing a microwell structure including a fluid storage cavity, a film-forming cavity and a connecting channel that penetrates a lower material layer on the substrate by exposure and development; and preparing a channel structure that penetrates an upper material layer on the lower material layer having the microwell structure by exposure and development.

[0070] It should be noted that the substrate in this embodiment is a mature product in the prior art, and the exposure and development technology are prior art, which will not be described in detail here. The upper and lower material layers of the film-forming microstructure in this application are made of the same material, such as SU-8 photoresist spin coating or dry film lamination. The following is a brief introduction to the preparation process using dry film as an example:

[0071] First, a lower dry film of moderate thickness is laminated on a substrate, and a photomask of the lower material layer is used for exposure and development, thereby obtaining a micro-well structure of a fluid storage cavity, a film-forming cavity and a connecting channel that penetrates the lower material layer;

[0072] Secondly, the dry film of the upper material layer is closely attached to the lower material layer with the micro-well structure, and a pore structure penetrating the upper material layer is prepared by exposure and development.

[0073] In summary, through the fluid storage chamber 23 connected to the film-forming chamber 22 provided in the present application, the size of the film-forming chamber 22 and the pore structure 11 can be designed to be smaller, so the actual film-forming area of ​​the amphiphilic molecular membrane will be smaller, and its stability will be further improved. Furthermore, since the fluid storage chamber and the film-forming chamber are relatively independent, they have greater flexibility in the size design of each, and a smaller film-forming chamber can be realized, which can achieve a better fixation effect on the molecular film, reduce the actual film-forming area, and increase the stability of the film; to a certain extent, it solves the contradiction between the small-sized micro-support structure required for the stability of the molecular film and the large volume required by the sequencing reaction system, gets rid of the size constraints between the two, and increases the flexibility of the design.

[0074] The above description shows and describes the preferred embodiments of the present application, but as mentioned above, it should be understood that the present application is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the object concept of the present application through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present application, and should be protected by the claims attached to the present application.

Claims

1. A film-forming microstructure for nanopore gene sequencing, comprising a micro-support structure unit arranged on a substrate, characterized in that: The micro-support structure unit includes a support cavity structure arranged on an upper material layer and a micro-well structure corresponding to the support cavity structure arranged on a lower material layer, wherein the support cavity structure includes a pore structure; The micro-well structure includes a film-forming cavity which is arranged correspondingly to the pore structure, and a fluid storage cavity which is connected to the film-forming cavity. The fluid storage cavity runs through the lower material layer, and at least one connecting channel is provided between the film-forming cavity and the fluid storage cavity.

2. The film-forming microstructure for nanopore gene sequencing according to claim 1, characterized in that: The cross-sectional shape of the fluid storage chamber includes one or more of a circle, an arc, an annular shape or a polygonal shape.

3. The film-forming microstructure for nanopore gene sequencing according to claim 1, characterized in that: The fluid in the fluid storage chamber is in direct contact with the detection electrode of the substrate, thereby realizing the transmission and collection of sequencing electrical signals.

4. The film-forming microstructure for nanopore gene sequencing according to claim 1, characterized in that: The film-forming cavity may or may not penetrate the lower material layer, but does not directly contact the detection electrode of the substrate.

5. The film-forming microstructure for nanopore gene sequencing according to claim 1, characterized in that: A plurality of exhaust holes are provided on the upper material layer above the fluid storage cavity to assist in exhausting the gas in the fluid storage cavity.

6. The film-forming microstructure for nanopore gene sequencing according to claim 1, characterized in that: The cross-sectional shape of the connecting channel is a straight line shape with a uniform opening from the fluid storage chamber to the film-forming chamber; or a gradually enlarging duckbill shape; or a dumbbell shape with a small middle and large ends.

7. The film-forming microstructure for nanopore gene sequencing according to claim 1, characterized in that: The width of the pore structure is D1, the width of the film-forming cavity is D2, and the width of the fluid storage cavity is D3. The relationship between them is D1 <D2<D3。 8. The film-forming microstructure for nanopore gene sequencing according to claim 7, characterized in that: The ratio of the width D1 of the pore structure to the width D2 of the film-forming cavity is between 1:1.5 and 1:

10.

9. The film-forming microstructure for nanopore gene sequencing according to claim 1, characterized in that: The ratio between the width D2 and the depth H2 of the film-forming cavity satisfies D2 / H2≥1:3, and the depth H2 of the film-forming cavity ranges from 50 μm to 1000 μm.

10. The film-forming microstructure for nanopore gene sequencing according to claim 1, characterized in that: The ratio between the width D3 and the depth H3 of the fluid storage chamber is in the range of 1:1 to 1:

10.

11. The film-forming microstructure for nanopore gene sequencing according to claim 1, characterized in that: The width w of the connecting channel ranges from 1 to 100 μm; the depth of the connecting channel is between the depth of the film-forming cavity and the depth of the fluid storage cavity.

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