Nanopore Detection Device and Method Based on Molecular Sealing Layer and Heating Sealing Structure

Abstract

The present invention provides a nanopore detection device and a manufacturing method based on a molecular sealing layer and a heating sealing structure. The device includes: a barrier layer formed with a plurality of nanopores and having a common liquid chamber above; a cavity layer including a plurality of independent cavities; a microchannel structure for injecting a solution; an oil-phase liquid sealing layer that forms an oil-water interface with the aqueous reaction solution in the independent cavities to seal and isolate the aqueous reaction solution in their respective independent cavities; a lipid molecular sealing layer whose hydrophilic groups are dissolved in the aqueous reaction solution and whose hydrophobic groups are dissolved in the oil-phase liquid sealing layer; a heating sealing structure for forming an air-sealed chamber at the bottom of the independent cavities. The present invention seals and isolates the aqueous reaction solution in their respective independent cavities through the oil-phase liquid sealing layer and the lipid molecular sealing layer, achieving the "double liquid sealing" effect of the independent cavities, and forms an air-sealed chamber at the bottom of the independent cavities through a heating electrode, achieving a good liquid sealing effect.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection devices and manufacturing, and particularly relates to a nanopore detection device, a manufacturing method and an application based on a molecular sealing layer and a heating sealing structure. Background Art

[0002] Most of the current nanopore sequencing technologies use the form of measuring ionic current to measure the blocking current generated by DNA passing through the pore. According to the differences in the size information and charge information of different bases, the size of the blocking current is different. Therefore, different bases correspond to different blocking currents, and thus the sequence information of DNA can be analyzed. Generally, the nanopore is on an insulating thin film. For example, a biological nanopore is embedded in an insulating lipid bilayer thin film, and a solid-state nanopore is prepared on a solid insulating thin film through semiconductor processing technology. The nanopore and the insulating thin film are placed in an electrolyte solution (generally a KCl solution), which divides the solution into two parts. A driving voltage is applied on both sides of the insulating thin film. This driving voltage has two functions: on the one hand, the voltage drives the charged ions in the salt solution to pass through the nanopore, and the movement of the charged ions generates an ionic current passing through the pore; on the other hand, the driving voltage drives the charged DNA molecules to move through the nanopore. When the DNA molecule moves in the nanopore, it blocks the movement of the ions in the nanopore. Therefore, the intensity of the ionic current will decrease, forming a blocking current. Since the size and charge information of the four bases of DNA are all different, the sizes of the blocking currents generated by different bases are different. This is the basic principle of nanopore sequencing.

[0003] If it is necessary to improve the sequencing throughput, a large number of nanopores need to carry out sequencing simultaneously. The nanopores are often prepared on a nanopore array chip. The nanopore array chip can share a solution system and a common electrode, but each nanopore also needs an independent electrode and an independent solution chamber, and there needs to be sufficient sealing conditions between these independent electrodes and solution chambers. The salt solution between the independent chambers cannot leak, otherwise leakage current and crosstalk phenomena will occur in the ionic current signals generated in each nanopore, resulting in adverse phenomena such as increased signal noise and cross-interference, affecting the accurate interpretation of the signals. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a nanopore detection device, a manufacturing method and an application based on a molecular sealing layer and a heating sealing structure, which are used to solve the problem that the ionic current signals generated in each nanopore of the nanopore array in the prior art are prone to leakage current and crosstalk.

[0005] To achieve the above and other related objectives, the present invention provides a nanopore detection device based on a molecular sealing layer and a heating sealing structure. The detection device includes: a barrier layer in which a plurality of nanopores penetrating the barrier layer are formed, and a common liquid chamber is provided above the barrier layer; a cavity layer located below the barrier layer, including a plurality of independent cavities, and each of the independent cavities is correspondingly configured with the nanopore; a microchannel structure located below the cavity layer, for injecting an aqueous reaction solution into the independent cavities, and injecting an oil-phase liquid sealing layer and a lipid molecular sealing layer onto the lower surface of the cavity layer; an oil-phase liquid sealing layer located on the lower surface of the cavity layer, and the oil-phase liquid sealing layer forms an oil-water interface with the aqueous reaction solution in the independent cavities to seal and isolate the aqueous reaction solution in their respective independent cavities; a lipid molecular sealing layer located at the oil-water interface, and the lipid molecular sealing layer includes a hydrophilic group and a hydrophobic group, the hydrophilic group is dissolved in the aqueous reaction solution, and the hydrophobic group is dissolved in the oil-phase liquid sealing layer; a heating sealing structure including a heating electrode located at the bottom of the independent cavity, for forming an air-sealed cavity at the bottom of the independent cavity.

[0006] Optionally, the nanopore includes one of a solid-state nanopore and a biological nanopore. The barrier layer of the solid-state nanopore includes an insulating dielectric layer, and the barrier layer of the biological nanopore includes one of a lipid molecular layer and a block copolymer molecular layer. The insulating dielectric layer includes one of silicon nitride, silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide, and graphene. The lipid molecular layer includes a phospholipid bilayer.

[0007] Optionally, the shape of the solid-state nanopore includes one of a cylindrical shape, a conical shape, a tower shape, and a funnel shape.

[0008] Optionally, the minimum pore diameter of the nanopore is 0.1 - 99 nm.

[0009] Optionally, the multiple nanopores in the barrier layer and the multiple independent cavities in the cavity layer are both arranged in a periodic array.

[0010] Optionally, it further includes an electrode structure, and the electrode structure includes a common electrode disposed in the common liquid chamber and independent electrodes disposed in each of the independent cavities.

[0011] Optionally, the independent cavity is a cylindrical cavity, the diameter of the cylindrical cavity is 1 - 1000 μm, and the interval between two adjacent cylindrical cavities is 2 - 5000 μm.

[0012] Optionally, the lipid molecular sealing layer includes one of phospholipid molecules, glycolipid molecules, diglycerides, triglycerides, and glycerophosphates.

[0013] Optionally, the hydrophilic group includes one of a hydroxyl group, a carboxyl group, an amino group, and a phosphate group, and the hydrophobic group includes an alkane chain.

[0014] Optionally, the heating electrode includes an annular heating electrode surrounding the bottom of the independent cavity.

[0015] Optionally, the detection device is used for detecting a DNA sequence. By applying a driving voltage on both sides of the nanopore, the ion movement in the aqueous reaction solution is driven to generate a current, and at the same time, the DNA strand is driven to pass through the nanopore. When the DNA strand passes through the nanopore, it blocks the ion movement, forming a blocking current. According to the correspondence between the blocking current and the DNA sequence, the DNA sequence is determined by measuring the magnitude of the blocking current.

[0016] The present invention also provides an application method of a nanopore detection device based on a molecular sealing layer and a heating and sealing structure, including: 1) injecting an aqueous reaction solution into the independent cavity based on the microchannel structure; 2) injecting an oil-phase liquid sealing layer dissolved with lipid molecules onto the lower surface of the cavity layer based on the microchannel structure. The oil-phase liquid sealing layer and the aqueous reaction solution in the independent cavity form an oil-water interface, and the lipid molecules self-assemble at the oil-water interface to form a lipid molecule sealing layer. The lipid molecule sealing layer includes a hydrophilic group and a hydrophobic group. The hydrophilic group is dissolved in the aqueous reaction solution, and the hydrophobic group is dissolved in the oil-phase liquid sealing layer to seal and isolate the aqueous reaction solution in their respective independent cavities; 3) heating through the heating electrode to form an air-sealed cavity at the bottom of the independent cavity; 4) by applying a driving voltage on both sides of the nanopore, the ion movement in the aqueous reaction solution is driven to generate a current, and at the same time, the DNA strand in the aqueous reaction solution is driven to pass through the nanopore. When the DNA strand passes through the nanopore, it blocks the ion movement, forming a blocking current. According to the correspondence between the blocking current and the DNA sequence, the DNA sequence is determined by measuring the magnitude of the blocking current.

[0017] The present invention also provides a method for manufacturing a nanopore detection device based on a molecular sealing layer and a heating and sealing structure. The manufacturing method includes the steps of: 1) providing a substrate, forming a dielectric layer on the substrate, and forming a barrier layer on the dielectric layer; 2) etching the substrate to form a common liquid cavity; 3) etching the dielectric layer to form a plurality of independent cavities in the dielectric layer to form a cavity layer; 4) forming a heating and sealing structure at the bottom of the independent cavity, the heating and sealing structure including a heating electrode located at the bottom of the independent cavity for forming an air-sealed cavity at the bottom of the independent cavity; 5) forming nanopores in the barrier layer, each independent cavity corresponding to one nanopore; 6) forming a microchannel structure below the cavity layer, the microchannel structure being used for injecting an aqueous reaction solution into the independent cavity and injecting an oil-phase liquid sealing layer onto the lower surface of the cavity layer; 7) forming an oil-phase liquid sealing layer dissolved with lipid molecules on the lower surface of the cavity layer, the oil-phase liquid sealing layer and the aqueous reaction solution in the independent cavity forming an oil-water interface, and the lipid molecules self-assembling at the oil-water interface to form a lipid molecule sealing layer, the lipid molecule sealing layer including a hydrophilic group and a hydrophobic group, the hydrophilic group being dissolved in the aqueous reaction solution, and the hydrophobic group being dissolved in the oil-phase liquid sealing layer to seal and isolate the aqueous reaction solution in their respective independent cavities.

[0018] Optionally, it further includes the step of preparing an electrode structure, the electrode structure including a common electrode disposed in the common liquid cavity and an independent electrode disposed in each independent cavity.

[0019] Optionally, the nanopore in step 5) includes one of a solid-state nanopore and a biological nanopore. The barrier layer of the solid-state nanopore includes an insulating dielectric layer, and the barrier layer of the biological nanopore includes one of a lipid molecule layer and a block copolymer molecule layer. The insulating dielectric layer includes one of silicon nitride, silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide, and graphene. The lipid molecule layer includes a phospholipid bilayer.

[0020] Optionally, the method for forming a solid-state nanopore in the barrier layer includes the steps of: forming a conductive metal in the independent cavity; forming an independent electrode corresponding to each independent cavity on the conductive metal, the independent electrode exposing a part of the independent cavity to form a removal window, making a common electrode in the common liquid cavity, the melting temperatures of the independent electrode and the common electrode being higher than the melting temperature of the conductive metal; applying a breakdown voltage between the independent electrode and the common electrode to break down the conductive metal through the barrier layer to simultaneously form nanopores corresponding to each independent cavity in the barrier layer; removing the conductive metal from the removal window by heating and melting.

[0021] Optionally, the conductive metal includes one of cadmium, tin, indium, and bismuth, and the materials of the independent electrode and the common electrode include one of copper, aluminum, titanium nitride, gold, and platinum.

[0022] Optionally, the shape of the solid-state nanopore includes one of cylindrical, conical, tower-shaped, and funnel-shaped.

[0023] Optionally, the lipid molecule seal layer includes one of phospholipid molecules, glycolipid molecules, diglycerides, triglycerides, and glycerophosphates.

[0024] Optionally, the hydrophilic group includes one of hydroxyl, carboxyl, amino, and phosphate groups, and the hydrophobic group includes an alkane chain.

[0025] As described above, the nanopore detection device, manufacturing method, and application based on the molecular seal layer and the heat-sealing structure of the present invention have the following beneficial effects:

[0026] The present invention provides a nanopore detection device based on a molecular seal layer and a heat-sealing structure. After the aqueous solution fills the common liquid chamber and the independent chamber, an oil-phase liquid seal layer is injected through a microchannel. The oil-phase liquid seal layer will squeeze out and replace the aqueous solution in the microchannel, covering the lower surface of the cavity layer. Under the action of surface tension, the aqueous solution in the independent chamber will not be replaced by the oil-phase liquid seal layer but will be enclosed in the independent chamber by the oil-phase liquid seal layer, forming an oil-water interface, sealing and isolating the aqueous reaction solution in their respective independent chambers, preventing possible cross-leakage of salt solutions between the independent chambers, and achieving the effect of sealing the independent chambers.

[0027] The present invention self-assembles a lipid molecule seal layer at the oil-water interface. The presence of the lipid molecule seal layer will enhance the liquid seal effect, and it, together with the oil-phase liquid seal layer, realizes the "double-layer liquid seal" of the independent chamber, further avoiding liquid leakage during the reaction process and achieving a better effect of sealing the liquid chamber for the independent chamber.

[0028] The present invention forms a heating electrode at the bottom of the independent chamber and heats the bottom of the independent chamber after being energized to form an air-sealed chamber at the bottom of the independent chamber, achieving an excellent liquid seal effect together with the oil-phase liquid seal layer and the lipid molecule seal layer.

[0029] The present invention forms a conductive metal in the independent chamber and simultaneously forms nanopores corresponding to each independent chamber in the barrier layer by applying a breakdown voltage, and then removes the conductive metal by heating and melting. On the one hand, it can realize the preparation of a nanopore array with high alignment accuracy, and on the other hand, it can effectively reduce the preparation cost of the nanopore array, having the advantages of simple and stable processes. Description of the Drawings

[0030] Figure 1It shows a schematic structural diagram of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to an embodiment of the present invention.

[0031] Figures 2 to 5 It shows a schematic diagram of a nanopore implementation manner of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to an embodiment of the present invention.

[0032] Figure 6 and Figure 7 It shows a schematic structural diagram of a lipid molecular sealing layer of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to an embodiment of the present invention.

[0033] Figure 8 It shows a schematic flow chart of the application method steps of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to an embodiment of the present invention.

[0034] Figure 9 It shows a schematic flow chart of the manufacturing method steps of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to an embodiment of the present invention.

[0035] Description of component labels

[0036] 101 Cavity layer

[0037] 102 Independent cavity

[0038] 103 Barrier layer

[0039] 104 Nanopore

[0040] 105 Microchannel structure

[0041] 106 Oil-phase liquid sealing layer

[0042] 107 Oil-water interface

[0043] 108 Common liquid cavity

[0044] 109 Independent electrode

[0045] 110 Common electrode

[0046] 111 Lipid molecular sealing layer

[0047] 1111 Hydrophilic group

[0048] 1112 Hydrophobic group

[0049] 112 Heating electrode

[0050] 113 Air-sealed cavity

[0051] Steps S11 to S13

[0052] Steps S21 to S26 Detailed implementation manners

[0053] The following uses specific specific examples to illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0054] When detailing the embodiments of the present invention, for ease of explanation, the cross-sectional views showing the device structure will be enlarged locally in a non-general proportion, and the schematic diagrams are only examples and should not limit the scope of protection of the present invention here. In addition, in actual production, three-dimensional spatial dimensions including length, width, and depth should be included.

[0055] For convenience of description, spatial relationship terms such as "beneath", "below", "lower than", "under", "above", "on" etc. may be used here to describe the relationship between one element or feature shown in the drawings and other elements or features. It will be understood that these spatial relationship terms are intended to include other directions of the device in use or operation besides the directions depicted in the drawings. In addition, when a layer is referred to as being "between" two layers, it can be the only layer between the two layers, or there can be one or more intervening layers.

[0056] In the context of the present application, the structure in which the first feature is "above" the second feature described may include an embodiment where the first and second features are formed in direct contact, and may also include an embodiment where additional features are formed between the first and second features, such that the first and second features may not be in direct contact.

[0057] It should be noted that the diagrams provided in this embodiment only illustrate the basic concept of the present invention in a schematic manner. Therefore, only the components related to the present invention are shown in the diagrams, rather than being drawn according to the number, shape, and size of the components in actual implementation. The types, quantities, and proportions of the components in actual implementation can be arbitrarily changed, and the component layout type may also be more complex.

[0058] As Figures 1 to 7 shown, this embodiment provides a nanopore detection device based on a molecular sealing layer and a heating sealing structure. The detection device includes: a common liquid chamber 108, a barrier layer 103, a cavity layer 101, a microchannel structure 105, an oil-phase liquid sealing layer 106, and a lipid molecular sealing layer 111.

[0059] As Figure 1 shown, a plurality of nanopores 104 penetrating through the barrier layer 103 are formed in the barrier layer 103.

[0060] The nanopore 104 includes one of a solid-state nanopore and a biological nanopore. The barrier layer 103 of the solid-state nanopore includes an insulating dielectric layer, and the barrier layer 103 of the biological nanopore includes one of a lipid molecule layer and a block copolymer molecule layer. The insulating dielectric layer includes one of silicon nitride, silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide, and graphene. The lipid molecule layer includes a phospholipid bilayer. In this embodiment, the nanopore 104 is a solid-state nanopore, and the barrier layer 103 is a silicon nitride layer.

[0061] As Figure 2 shown, in one embodiment, the shape of the solid-state nanopore is cylindrical, and the diameter of the nanopore 104 can be 0.1 - 99 nm. Preferably, the diameter of the nanopore 104 is 1 - 5 nm.

[0062] As Figure 3 shown, in another embodiment, the shape of the solid-state nanopore is conical. The conical nanopore 104 has a minimum pore diameter, which can be 0.1 - 99 nm. Preferably, the minimum pore diameter is 1 - 5 nm. Setting the solid-state nanopore to be conical can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore (i.e., the thickness of the barrier layer 103 corresponding to the minimum pore diameter is smaller), avoid the nanopore 104 being completely blocked while ensuring the measurement accuracy, and ensure the flow of DNA in the aqueous reaction solution. On the other hand, it can reduce the actual moving distance of DNA in the nanopore 104 and improve the detection accuracy.

[0063] As Figure 4 shown, in yet another embodiment, the shape of the solid-state nanopore is tower-shaped. The tower-shaped nanopore 104 is formed by sequentially connecting two or more circular holes with different diameters. The tower-shaped nanopore 104 has a minimum pore diameter, which can be 0.1 - 99 nm. Preferably, the minimum pore diameter is 1 - 5 nm. Setting the solid-state nanopore to be tower-shaped can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore (i.e., the thickness of the barrier layer 103 corresponding to the minimum pore diameter is smaller), avoid the nanopore 104 being completely blocked while ensuring the measurement accuracy, and ensure the flow of DNA in the aqueous reaction solution. On the other hand, it can reduce the actual moving distance of DNA in the nanopore 104 and improve the detection accuracy.

[0064] As Figure 5As shown, in another embodiment, the shape of the solid-state nanopore is funnel-shaped. The funnel-shaped nanopore 104 is formed by connecting two opposite conical holes. The funnel-shaped nanopore 104 has a minimum pore diameter, which can be 0.1 to 99 nm, preferably 1 to 5 nm. Setting the solid-state nanopore to be funnel-shaped can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore (i.e., the thickness of the barrier layer 103 corresponding to the minimum pore diameter is smaller), avoid the complete blockage of the nanopore 104 while ensuring the measurement accuracy, and ensure the flow of DNA in the aqueous reaction solution. On the other hand, it can reduce the actual moving distance of DNA in the nanopore 104 and improve the detection accuracy.

[0065] As Figure 1 shown, the common liquid chamber 108 is located above the barrier layer 103 and is used to carry the aqueous reaction solution. The aqueous reaction solution in the common liquid chamber 108 can be directly injected or injected through a microchannel structure.

[0066] As Figure 1 shown, the cavity layer 101 is located below the barrier layer 103. The cavity layer 101 includes a plurality of independent cavities 102, and each independent cavity 102 is correspondingly configured with a nanopore 104. The material of the cavity layer 101 can be silicon dioxide, etc. The plurality of independent cavities 102 are etched in the silicon dioxide by means of photolithography-etching. The independent cavity 102 can be a cylindrical cavity, and the diameter of the cylindrical cavity is 1 to 1000 μm, and the interval between adjacent two cylindrical cavities is 2 to 5000 μm. Of course, in other embodiments, the shape of the independent cavity can also be other shapes such as oval, polygon, etc., and is not limited to the examples listed here.

[0067] In this embodiment, the plurality of nanopores 104 in the barrier layer 103 and the plurality of independent cavities 102 in the cavity layer 101 are both arranged in a periodic array to improve the detection throughput and efficiency.

[0068] As Figure 1 shown, the microchannel structure 105 is located below the cavity layer 101 and is used to inject the aqueous reaction solution into the independent cavity 102, and inject the oil-phase liquid seal layer 106 and the lipid molecule seal layer 111 onto the lower surface of the cavity layer 101.

[0069] As Figure 1As shown, the oil-phase liquid seal layer 106 is located on the lower surface of the cavity layer 101. The oil-phase liquid seal layer 106 and the aqueous reaction solution in the independent cavity 102 form an oil-water interface 107 to enclose and isolate the aqueous reaction solution in their respective independent cavities 102. After the aqueous solution fills the common liquid cavity 108 and the independent cavities 102, the oil-phase liquid seal layer 106 is injected through the microchannel. The oil-phase liquid seal layer 106 will cover the lower surface of the cavity layer 101. As Figure 1 shown, under the action of surface tension, the aqueous solution in the independent cavity 102 will not be replaced by the oil-phase liquid seal layer 106, but will be enclosed in the independent cavity 102 by the oil-phase liquid seal layer 106 to form an oil-water interface 107, enclosing and isolating the aqueous reaction solution in their respective independent cavities 102, preventing possible cross-leakage of salt solutions between the independent cavities 102, and achieving the effect of sealing the independent cavities 102.

[0070] As Figure 6 and Figure 7 shown, the lipid molecule seal layer 111 is located at the oil-water interface 107. The lipid molecule seal layer 111 includes a hydrophilic group 1111 and a hydrophobic group 1112. The hydrophilic group 1111 is dissolved in the aqueous reaction solution, and the hydrophobic group 1112 is dissolved in the oil-phase liquid seal layer. The lipid molecule seal layer 111 includes amphiphilic molecules. When amphiphilic molecules are dissolved alone in an aqueous solution, the amphiphilic molecules will slowly undergo self-assembly behavior on the surface of the aqueous solution, with their hydrophilic groups 1111 dissolved in water and their hydrophobic groups 1112 distributed in the air; when amphiphilic molecules are dissolved alone in an oil-phase organic solvent, the same self-assembly behavior will occur, with their hydrophobic groups 1112 (such as alkane chains) dissolved in the oil phase and their hydrophilic groups 1111 distributed in the air; in a mixed liquid of an oil phase and a water phase, the amphiphilic molecules will undergo self-assembly behavior at the oil-water interface, with their hydrophilic groups 1111 dissolved in the aqueous solution and their hydrophobic groups 1112 dissolved in the oil-phase solvent. In this embodiment, the lipid molecule seal layer includes one of phospholipid molecules, glycolipid molecules, diglycerides, triglycerides, and glycerophosphates. As an example, as Figure 7 shown, the hydrophilic group 1111 includes one of a hydroxyl group, a carboxyl group, an amino group, and a phosphate group, and the hydrophobic group 1112 includes an alkane chain. In this embodiment, the number of alkane chains is 1 to 3.

[0071] In the present invention, a lipid molecule seal layer is self-assembled at the oil-water interface. The presence of the lipid molecule seal layer will enhance the liquid seal effect, and it and the oil-phase liquid seal layer jointly achieve "double-layer liquid sealing" of the independent cavity, further avoiding liquid leakage during the reaction process and achieving a better effect of sealing the liquid cavity of the independent cavity.

[0072] As Figure 1As shown, the heating and sealing structure includes a heating electrode 112 located at the bottom of the independent cavity 102, which is used to form an air-sealed cavity 113 at the bottom of the independent cavity 102.

[0073] The heating electrode 112 includes an annular heating electrode surrounding the bottom of the independent cavity 102. In this embodiment, the heating electrode 112 is an annular heating electrode surrounding the bottom of the independent cavity 102, which is beneficial to the formation of the air-sealed cavity 113 and is easy to form an air-sealed cavity 113 that completely covers the entire bottom of the independent cavity 102, and can very effectively achieve the sealing and isolation of the independent cavity 102. Of course, in other embodiments, the heating electrode 112 can also be a block electrode, and two or more block electrodes are evenly distributed at the bottom of the independent cavity 102 to form an air-sealed cavity 113 at the bottom of the independent cavity 102. It should be noted that the structure of the heating electrode 112 is not limited to the examples listed above and can be selected according to actual needs. As Figure 1 As shown, the detection device further includes an electrode structure, which includes a common electrode 110 disposed in the common liquid cavity 108 and an independent electrode 109 disposed in each independent cavity 102.

[0074] The detection device is used for DNA sequence detection. By applying a driving voltage across both sides of the nanopore 104, the ions in the aqueous reaction solution are driven to move to generate a current, and at the same time, the DNA strand is driven to pass through the nanopore 104. When the DNA strand passes through the nanopore 104, it blocks the ion movement to form a blocking current. According to the corresponding relationship between the blocking current and the DNA sequence, the DNA sequence is determined by measuring the magnitude of the blocking current.

[0075] As Figure 8 As shown, the present invention also provides an application method of a nanopore detection device based on a molecular sealing layer and a heating and sealing structure, including:

[0076] Step 1) S11: Inject an aqueous reaction solution containing the DNA to be detected and a dielectric solution (such as potassium chloride (KCl) solution) into the independent cavity 102 based on the microchannel structure 105.

[0077] Step 2) S12: Inject the oil-phase liquid seal layer 106 dissolved with lipid molecules onto the lower surface of the cavity layer 101 based on the microchannel structure 105. An oil-water interface 107 is formed between the oil-phase liquid seal layer 106 and the aqueous reaction solution in the independent cavity 102. The lipid molecules self-assemble at the oil-water interface to form a lipid molecule seal layer 111. The lipid molecule seal layer 111 includes a hydrophilic group 1111 and a hydrophobic group 1112. The hydrophilic group 1111 is dissolved in the aqueous reaction solution, and the hydrophobic group 1112 is dissolved in the oil-phase liquid seal layer 106, so as to seal and isolate the aqueous reaction solution in their respective independent cavities 102, achieving the effect of "double-layer liquid seal".

[0078] Step 3) S13: Heat through the heating electrode 112 to form an air closed cavity 113 at the bottom of the independent cavity 102. The air closed cavity 113 completely covers the bottom of the independent cavity 102.

[0079] Step 4) S14: Apply a driving voltage on both sides of the nanopore 104 to drive the movement of ions in the aqueous reaction solution to generate a current, and at the same time drive the DNA strand in the aqueous reaction solution to pass through the nanopore 104. When the DNA strand passes through the nanopore 104, it blocks the movement of the ions, forming a blocking current. According to the correspondence between the blocking current and the sequence of the DNA, determine the sequence of the DNA by measuring the magnitude of the blocking current.

[0080] As Figures 1 to 7 and Figure 9 shown, the present invention also provides a manufacturing method of a nanopore detection device based on a molecular seal layer and a heating seal structure. The manufacturing method includes the steps:

[0081] As Figure 1 and Figure 9 shown, first perform Step 1) S21: Provide a substrate, form a dielectric layer on the substrate, and form a barrier layer 103 on the dielectric layer.

[0082] In this embodiment, the substrate is a silicon substrate, the dielectric layer is a silicon dioxide layer, the barrier layer 103 includes an insulating dielectric layer or a lipid molecule layer. The insulating dielectric layer includes one of silicon nitride, silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide, and graphene. The lipid molecule layer includes a phospholipid bilayer. Specifically, according to the different nanopores 104 formed subsequently, the nanopore 104 includes one of a solid-state nanopore and a biological nanopore. The barrier layer 103 of the solid-state nanopore includes an insulating dielectric layer, and the barrier layer 103 of the biological nanopore includes one of a lipid molecule layer and a block copolymer molecule layer.

[0083] As Figure 1 and Figure 9 shown, then step 2) S22 is performed to etch the substrate to form a common liquid cavity 108.

[0084] As Figure 1 and Figure 9 shown, then step 3) S23 is performed to etch the dielectric layer to form a plurality of independent cavities 102 in the dielectric layer to form a cavity layer 101.

[0085] The material of the cavity layer 101 may be silicon dioxide or the like. The plurality of independent cavities 102 are etched in the silicon dioxide by means of photolithography - etching. The independent cavities 102 may be cylindrical cavities, and the diameter of the cylindrical cavities is 1 - 1000 μm, and the interval between two adjacent cylindrical cavities is 2 - 5000 μm. Of course, in other embodiments, the shape of the independent cavity may also be an ellipse, a polygon or other shapes, and is not limited to the examples listed here.

[0086] In this embodiment, the plurality of nanopores 104 in the barrier layer 103 and the plurality of independent cavities 102 in the cavity layer 101 are both arranged in a periodic array to improve the detection throughput and efficiency.

[0087] As Figure 1 and Figure 9 shown, then step 4) S24 is performed to form a heating and sealing structure at the bottom of the independent cavity 102. The heating and sealing structure includes a heating electrode 112 located at the bottom of the independent cavity 102 for forming an air - closed cavity 113 at the bottom of the independent cavity 102.

[0088] In this embodiment, the heating electrode 112 includes a ring - shaped heating electrode surrounding the bottom of the independent cavity 102. The heating electrode 112 in this embodiment adopts a ring - shaped heating electrode surrounding the bottom of the independent cavity 102, which is beneficial to the formation of the air - closed cavity 113 and is easy to form an air - closed cavity 113 that completely covers the entire bottom of the independent cavity 102, and can very effectively achieve the sealing and isolation of the independent cavity 102. Of course, in other embodiments, the heating electrode 112 may also adopt a block - shaped electrode, and two or more block - shaped electrodes are uniformly distributed at the bottom of the independent cavity 102 to form an air - closed cavity 113 at the bottom of the independent cavity 102. It should be noted that the structure of the heating electrode 112 is not limited to the examples listed above and can be selected according to actual needs.

[0089] As Figure 1 and Figure 9As shown, then step 5) S25 is carried out to form nanopores 104 in the barrier layer 103, and each of the independent cavities 102 is correspondingly provided with the nanopores 104.

[0090] The nanopores 104 include one of solid-state nanopores and biological nanopores. In this embodiment, the nanopores 104 are solid-state nanopores, and the shape of the solid-state nanopores includes one of cylindrical, conical, tower-shaped, and funnel-shaped.

[0091] As Figure 2 shown, in one embodiment, the shape of the solid-state nanopore is cylindrical, and the diameter of the nanopore 104 can be 0.1 - 99 nm. Preferably, the diameter of the nanopore 104 is 1 - 5 nm.

[0092] As Figure 3 shown, in another embodiment, the shape of the solid-state nanopore is conical. The conical nanopore 104 has a minimum pore diameter, and the minimum pore diameter can be 0.1 - 99 nm. Preferably, the minimum pore diameter is 1 - 5 nm. Setting the solid-state nanopore to be conical can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore (i.e., the thickness of the barrier layer 103 corresponding to the minimum pore diameter is smaller), avoid the nanopore 104 from being completely blocked while ensuring the measurement accuracy, and ensure the flow of DNA in the aqueous reaction solution. On the other hand, it can reduce the actual moving distance of DNA in the nanopore 104 and improve the detection accuracy.

[0093] As Figure 4 shown, in yet another embodiment, the shape of the solid-state nanopore is tower-shaped. The tower-shaped nanopore 104 is formed by sequentially connecting two or more circular holes with different diameters. The tower-shaped nanopore 104 has a minimum pore diameter, and the minimum pore diameter can be 0.1 - 99 nm. Preferably, the minimum pore diameter is 1 - 5 nm. Setting the solid-state nanopore to be tower-shaped can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore (i.e., the thickness of the barrier layer 103 corresponding to the minimum pore diameter is smaller), avoid the nanopore 104 from being completely blocked while ensuring the measurement accuracy, and ensure the flow of DNA in the aqueous reaction solution. On the other hand, it can reduce the actual moving distance of DNA in the nanopore 104 and improve the detection accuracy.

[0094] As Figure 5As shown, in yet another embodiment, the shape of the solid-state nanopore is funnel-shaped. The funnel-shaped nanopore 104 is formed by connecting two opposite conical holes. The funnel-shaped nanopore 104 has a minimum pore diameter, which can be 0.1 - 99 nm. Preferably, the minimum pore diameter is 1 - 5 nm. Setting the solid-state nanopore to be funnel-shaped can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore (i.e., the thickness of the barrier layer 103 corresponding to the minimum pore diameter is smaller), which can avoid the complete blockage of the nanopore 104 while ensuring the measurement accuracy and ensure the flow of DNA in the aqueous reaction solution. On the other hand, it can reduce the actual moving distance of DNA in the nanopore 104 and improve the detection accuracy.

[0095] In this embodiment, the method for forming a solid-state nanopore in the barrier layer 103 includes the steps:

[0096] Step 5-1): Form a conductive metal in the independent cavity 102; form an independent electrode 109 corresponding to each independent cavity 102 on the conductive metal. The independent electrode 109 exposes part of the independent cavity 102 to form a removal window. Make a common electrode 110 in the common liquid cavity 108. The melting temperatures of the independent electrode 109 and the common electrode 110 are greater than the melting temperature of the conductive metal;

[0097] Step 5-2): Apply a breakdown voltage between the independent electrode 109 and the common electrode to break down the conductive metal through the barrier layer 103, so as to simultaneously form nanopores 104 corresponding to each independent cavity 102 in the barrier layer 103;

[0098] Step 5-3): Remove the conductive metal from the removal window by heating and melting.

[0099] For example, the conductive metal includes one of cadmium, tin, indium, and bismuth, and the materials of the independent electrode 109 and the common electrode 110 include one of copper, aluminum, titanium nitride, gold, and platinum.

[0100] In the present invention, a conductive metal is formed in the independent cavity 102, and nanopores 104 corresponding to each independent cavity 102 are simultaneously formed in the barrier layer 103 by applying a breakdown voltage. Then, the conductive metal is removed by heating and melting. On the one hand, it can realize the preparation of a nanopore 104 array with high alignment accuracy. On the other hand, it can effectively reduce the preparation cost of the nanopore 104 array, and has the advantages of simple and stable processes.

[0101] Such as Figure 1 and Figure 9As shown, then step 6) S26 is carried out to form a microchannel structure 105 below the cavity layer 101. The microchannel structure 105 is used to inject the aqueous reaction solution into the independent cavity 102 and the common liquid cavity 108, and to inject the oil-phase liquid seal layer 106 onto the lower surface of the cavity layer 101.

[0102] As Figure 1 and Figure 9 shown, then step 7) S27 is carried out to form an oil-phase liquid seal layer 106 dissolved with lipid molecules on the lower surface of the cavity layer 101. The oil-phase liquid seal layer 106 and the aqueous reaction solution in the independent cavity 102 form an oil-water interface 107. The lipid molecules self-assemble at the oil-water interface to form a lipid molecule seal layer 111. The lipid molecule seal layer 111 includes a hydrophilic group 1111 and a hydrophobic group 1112. The hydrophilic group 1111 is dissolved in the aqueous reaction solution, and the hydrophobic group 1112 is dissolved in the oil-phase liquid seal layer 106 to enclose and isolate the aqueous reaction solution in their respective independent cavities 102.

[0103] As Figure 1 shown, under the action of surface tension, the aqueous solution in the independent cavity 102 will not be replaced by the oil-phase liquid seal layer 106, but is enclosed in the independent cavity 102 by the oil-phase liquid seal layer 106 to form an oil-water interface 107, enclosing and isolating the aqueous reaction solution in their respective independent cavities 102, preventing possible cross-leakage of salt solutions between the independent cavities 102, and achieving the effect of sealing the independent cavities 102.

[0104] The concentration range of lipid molecules dissolved in the oil-phase liquid seal layer 106 is 1 μM to 10 M. In this embodiment, the concentration range of lipid molecules dissolved in the oil-phase liquid seal layer 106 is 100 nM to 1000 mM.

[0105] As Figure 6 and Figure 7As shown, the lipid molecule seal layer 111 is located at the oil-water interface 107. The lipid molecule seal layer 111 includes a hydrophilic group 1111 and a hydrophobic group 1112. The hydrophilic group 1111 is dissolved in the aqueous reaction solution, and the hydrophobic group 1112 is dissolved in the oil-phase liquid seal layer. The lipid molecule seal layer 111 includes amphiphilic molecules. When amphiphilic molecules are dissolved alone in an aqueous solution, the amphiphilic molecules will slowly self-assemble on the surface of the aqueous solution. Its hydrophilic group 1111 is dissolved in water, and the hydrophobic group 1112 is distributed in the air; when amphiphilic molecules are dissolved alone in an oil-phase organic solvent, self-assembly behavior will also occur. Its hydrophobic group 1112 (such as an alkane chain) is dissolved in the oil phase, and the hydrophilic group 1111 is distributed in the air; in a mixed liquid of an oil phase and an aqueous phase, the amphiphilic molecules will self-assemble at the oil-water interface. The hydrophilic group 1111 is dissolved in the aqueous solution, and the hydrophobic group 1112 is dissolved in the oil-phase solvent. In this embodiment, the lipid molecule seal layer includes one of phospholipid molecules, glycolipid molecules, diglycerides, triglycerides, and glycerophosphates. As an example, as Figure 7 shown, the hydrophilic group 1111 includes one of a hydroxyl group, a carboxyl group, an amino group, and a phosphate group, and the hydrophobic group 1112 includes an alkane chain. In this embodiment, the number of alkane chains is 1 to 3.

[0106] The present invention self-assembles to form a lipid molecule seal layer at the oil-water interface. The presence of the lipid molecule seal layer will enhance the liquid seal effect. It and the oil-phase liquid seal layer jointly achieve the "double-layer liquid seal" of the independent cavity, further avoiding liquid leakage during the reaction process, and achieving a better effect of sealing the liquid cavity for the independent cavity.

[0107] As described above, the nanopore detection device, manufacturing method, and application based on the molecular seal layer and the heating seal structure of the present invention have the following beneficial effects:

[0108] The present invention provides a nanopore detection device based on a molecular seal layer and a heating seal structure. After the aqueous solution fills the common liquid cavity and the independent cavity, an oil-phase liquid seal layer is injected through the microchannel. The oil-phase liquid seal layer will squeeze out and replace the aqueous solution in the microchannel, covering the lower surface of the cavity layer. Under the action of surface tension, the aqueous solution in the independent cavity will not be replaced by the oil-phase liquid seal layer, but will be sealed in the independent cavity by the oil-phase liquid seal layer, forming an oil-water interface, sealing and isolating the aqueous reaction solution in their respective independent cavities, preventing possible cross-leakage of salt solutions between independent cavities, and achieving the effect of sealing the independent cavity.

[0109] The present invention self-assembles to form a lipid molecular sealing layer at the oil-water interface. The presence of the lipid molecular sealing layer enhances the liquid sealing effect. Together with the oil-phase liquid sealing layer, it realizes the "double-layer liquid sealing" of the independent cavity, further avoiding liquid leakage during the reaction process and achieving a better effect of sealing the liquid cavity for the independent cavity.

[0110] The present invention sets a heating electrode at the bottom of the independent cavity. After being energized, the bottom of the independent cavity is heated to form an air-sealed cavity at the bottom of the independent cavity, and together with the oil-phase liquid sealing layer and the lipid molecular sealing layer, it realizes an excellent liquid sealing effect.

[0111] The present invention forms a conductive metal in the independent cavity, and forms nano-pores corresponding to each independent cavity in the barrier layer by applying a breakdown voltage. Then, the conductive metal is removed by heating and melting. On the one hand, it can realize the preparation of a nano-pore array with high alignment accuracy. On the other hand, it can effectively reduce the preparation cost of the nano-pore array, and has the advantages of simple and stable processes.

[0112] Therefore, the present invention effectively overcomes various disadvantages in the prior art and has high industrial utilization value.

[0113] The above embodiments are only illustrative of the principles and effects of the present invention, and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A nanopore detection device based on a molecular sealing layer and a heating sealing structure, characterized in that, the detection device includes: a barrier layer, in which a plurality of nanopores penetrating the barrier layer are formed, the nanopores include one of solid-state nanopores and biological nanopores, and a common liquid chamber is provided above the barrier layer; a cavity layer, located below the barrier layer, including a plurality of independent cavities, and each of the independent cavities is correspondingly provided with the nanopores; a microchannel structure, located below the cavity layer, for injecting an aqueous reaction solution into the independent cavities, and injecting an oil-phase liquid sealing layer and a lipid molecular sealing layer onto the lower surface of the cavity layer; an oil-phase liquid sealing layer, located on the lower surface of the cavity layer, the oil-phase liquid sealing layer and the aqueous reaction solution in the independent cavities form an oil-water interface to seal and isolate the aqueous reaction solution in their respective independent cavities; a lipid molecular sealing layer, located at the oil-water interface, the lipid molecular sealing layer includes a hydrophilic group and a hydrophobic group, the hydrophilic group is dissolved in the aqueous reaction solution, and the hydrophobic group is dissolved in the oil-phase liquid sealing layer; a heating sealing structure, including a heating electrode located at the bottom of the independent cavity, for forming an air-sealed cavity at the bottom of the independent cavity; further including an electrode structure, the electrode structure includes a common electrode disposed in the common liquid chamber and independent electrodes disposed in each of the independent cavities.

2. The nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 1, characterized in that: the barrier layer of the solid-state nanopore includes an insulating dielectric layer, and the barrier layer of the biological nanopore includes one of a lipid molecular layer and a block copolymer molecular layer, the insulating dielectric layer includes one of silicon nitride, silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide and graphene, and the lipid molecular layer includes a phospholipid bilayer.

3. The nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 2, characterized in that: the shape of the solid-state nanopore includes one of a cylindrical shape, a conical shape, a tower shape and a funnel shape.

4. The nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 1, characterized in that: the minimum pore diameter of the nanopore is 1 to 5 nm.

5. The nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 1, characterized in that: the plurality of nanopores in the barrier layer and the plurality of independent cavities in the cavity layer are both arranged in a periodic array.

6. The nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 1, characterized in that: the independent cavity is a cylindrical cavity, the diameter of the cylindrical cavity is 1 to 1000 μm, and the interval between two adjacent cylindrical cavities is 2 to 5000 μm.

7. The nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 1, characterized in that: the lipid molecular sealing layer includes one of phospholipid molecules, glycolipid molecules, diglycerides, triglycerides and glycerophosphates.

8. The nanopore detection device based on a molecular seal layer and a heating and sealing structure according to claim 1, characterized in that: the hydrophilic group includes one of a hydroxyl group, a carboxyl group, an amino group, and a phosphate group, and the hydrophobic group includes an alkane chain.

9. The nanopore detection device based on a molecular seal layer and a heating and sealing structure according to claim 1, characterized in that: the heating electrode includes an annular heating electrode surrounding the bottom of the independent cavity.

10. The nanopore detection device based on a molecular seal layer and a heating and sealing structure according to claim 1, characterized in that: the detection device is used for detecting DNA sequences. By applying a driving voltage on both sides of the nanopore, the ions in the aqueous reaction solution are driven to move to generate a current, and at the same time, the DNA strand is driven to pass through the nanopore. When the DNA strand passes through the nanopore, it blocks the ion movement, forming a blocking current. According to the corresponding relationship between the blocking current and the DNA sequence, the DNA sequence is determined by measuring the magnitude of the blocking current.

11. An application method of the nanopore detection device based on a molecular seal layer and a heating and sealing structure according to any one of claims 1 to 10, characterized in that it includes: 1) Injecting an aqueous reaction solution into the independent cavity based on the microchannel structure; 2) Injecting an oil-phase liquid seal layer dissolved with lipid molecules onto the lower surface of the cavity layer based on the microchannel structure. The oil-phase liquid seal layer and the aqueous reaction solution in the independent cavity form an oil-water interface. The lipid molecules self-assemble at the oil-water interface to form a lipid molecular seal layer. The lipid molecular seal layer includes a hydrophilic group and a hydrophobic group. The hydrophilic group is dissolved in the aqueous reaction solution, and the hydrophobic group is dissolved in the oil-phase liquid seal layer to seal and isolate the aqueous reaction solution in their respective independent cavities; 3) Heating through the heating electrode to form an air-sealed cavity at the bottom of the independent cavity; 4) By applying a driving voltage on both sides of the nanopore, the ions in the aqueous reaction solution are driven to move to generate a current, and at the same time, the DNA strand in the aqueous reaction solution is driven to pass through the nanopore. When the DNA strand passes through the nanopore, it blocks the ion movement, forming a blocking current. According to the corresponding relationship between the blocking current and the DNA sequence, the DNA sequence is determined by measuring the magnitude of the blocking current.

12. A manufacturing method of a nanopore detection device based on a molecular seal layer and a heating and sealing structure, characterized in that the manufacturing method includes the steps: 1) Providing a substrate, forming a dielectric layer on the substrate, and forming a barrier layer on the dielectric layer; 2) Etching the substrate to form a common liquid cavity; 3) Etching the dielectric layer to form a plurality of independent cavities in the dielectric layer to form a cavity layer; 4) Forming a heating and sealing structure at the bottom of the independent cavity. The heating and sealing structure includes a heating electrode located at the bottom of the independent cavity for forming an air-sealed cavity at the bottom of the independent cavity; 5) Form nanopores in the barrier layer, with each independent cavity correspondingly configured with a nanopore; 6) Form a microchannel structure under the cavity layer, where the microchannel structure is used to inject an aqueous reaction solution into the independent cavity and to inject an oil-phase liquid seal layer onto the lower surface of the cavity layer; 7) Form an oil-phase liquid seal layer dissolved with lipid molecules on the lower surface of the cavity layer. The oil-phase liquid seal layer and the aqueous reaction solution in the independent cavity form an oil-water interface. The lipid molecules self-assemble to form a lipid molecule sealing layer at the oil-water interface. The lipid molecule sealing layer includes a hydrophilic group and a hydrophobic group. The hydrophilic group is dissolved in the aqueous reaction solution, and the hydrophobic group is dissolved in the oil-phase liquid seal layer to enclose and isolate the aqueous reaction solution in their respective independent cavities; It further includes the step of preparing an electrode structure, where the electrode structure includes a common electrode disposed in the common liquid cavity and independent electrodes disposed in each independent cavity.

13. The manufacturing method of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 12, characterized in that: In step 5), the nanopore includes a biological nanopore, and the barrier layer of the biological nanopore includes one of a lipid molecule layer and a block copolymer molecule layer, and the lipid molecule layer includes a phospholipid bilayer.

14. The manufacturing method of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 12, characterized in that: In step 5), the nanopore includes a solid-state nanopore, and the barrier layer of the solid-state nanopore includes an insulating dielectric layer, and the insulating dielectric layer includes one of silicon nitride, silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide, and graphene.

15. The manufacturing method of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 14, characterized in that: The method of forming a solid-state nanopore in the barrier layer includes the steps of: Form a conductive metal in the independent cavity; Form independent electrodes corresponding to each independent cavity on the conductive metal. The independent electrodes expose a part of the independent cavity to form a removal window, and a common electrode is fabricated in the common liquid cavity. The melting temperature of the independent electrodes and the common electrode is greater than the melting temperature of the conductive metal; By applying a breakdown voltage between the independent electrode and the common electrode, the conductive metal breaks through the barrier layer to simultaneously form nanopores corresponding to each independent cavity in the barrier layer; Remove the conductive metal from the removal window by heating and melting.

16. The manufacturing method of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 15, characterized in that: The conductive metal includes one of cadmium, tin, indium, and bismuth, and the materials of the independent electrodes and the common electrode include one of copper, aluminum, titanium nitride, gold, and platinum.

17. The manufacturing method of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 14, characterized in that: The shape of the solid-state nanopore includes one of cylindrical, conical, tower-shaped, and funnel-shaped.

18. The manufacturing method of the nanopore detection device based on the molecular seal layer and the heating sealing structure according to claim 12, characterized in that: The lipid molecular seal layer includes one of phospholipid molecules, glycolipid molecules, diglycerides, triglycerides, and glycerophosphates.

19. The manufacturing method of the nanopore detection device based on the molecular seal layer and the heating sealing structure according to claim 12, characterized in that: The hydrophilic group includes one of a hydroxyl group, a carboxyl group, an amino group, and a phosphate group, and the hydrophobic group includes an alkane chain.

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