Nanopore Detection Device, Manufacturing Method and Application Based on Heating Sealing Structure

Abstract

The present invention provides a nanopore detection device, a manufacturing method and an application based on a heating and sealing structure. The device includes: a barrier layer having a plurality of nanopores and having a common liquid chamber above; a cavity layer including a plurality of independent cavities, each independent cavity being correspondingly provided with a nanopore; a microchannel structure for injecting a solution; an oil-phase liquid sealing layer, the oil-phase liquid sealing layer forming an oil-water interface with the aqueous reaction solution in the independent cavity to seal and isolate the aqueous reaction solution in their respective independent cavities; a heating and sealing structure for forming an air-sealed cavity at the bottom of the independent cavity. The present invention seals and isolates the aqueous reaction solution in their respective independent cavities through the oil-phase liquid sealing layer, preventing possible cross-leakage of salt solutions between independent cavities and achieving the effect of sealing the independent cavities. At the same time, through the heating electrode, an air-sealed cavity is formed at the bottom of the independent cavity, jointly achieving a good liquid sealing effect with the oil-phase liquid sealing layer.

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 based on a heating and sealing structure, a manufacturing method and an application thereof. Background Art

[0002] Most of the current nanopore sequencing technologies measure the blockade current generated by DNA passing through the pore in the form of measuring ion current. According to the differences in the size information and charge information of different bases, the magnitude of the blockade current is different. Therefore, different bases correspond to different blockade currents, and thus the sequence information of DNA can be analyzed. Generally, the nanopore is on a layer of insulating film. For example, a biological nanopore is embedded in an insulating lipid bilayer film, and a solid-state nanopore is prepared on a solid insulating film through semiconductor processing technology. The nanopore and the insulating film are placed in an electrolyte solution (usually a KCl solution), and the solution is divided into two parts. A driving voltage is applied on both sides of the insulating 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 ion current passing through the pore; on the other hand, the driving voltage drives the charged DNA molecule 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 ion current will decrease, forming a blockade current. Since the sizes and charge information of the four bases of DNA are all different, the magnitudes of the blockade 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 ion 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 based on a heating and sealing structure, a manufacturing method and an application thereof, which are used to solve the problem that the ion 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 heating and 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 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 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.

[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 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.

[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 plurality of nanopores in the barrier layer and the plurality of 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 an independent electrode disposed in each independent cavity.

[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 heating electrode includes an annular heating electrode surrounding the bottom of the independent cavity.

[0013] Optionally, the detection device is used for detecting DNA sequences. By applying a driving voltage across the two 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.

[0014] The present invention also provides an application method of a nanopore detection device based on 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 seal layer onto the lower surface of the cavity layer based on the microchannel structure, and the oil-phase liquid seal layer and the aqueous reaction solution in the independent cavity form an oil-water interface 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) applying a driving voltage across the two sides of the nanopore to drive the ion movement in the aqueous reaction solution to generate a current, and at the same time, driving the DNA strand in the aqueous reaction solution 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.

[0015] The present invention also provides a manufacturing method of a nanopore detection device based on 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, and 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) forming a nanopore in the barrier layer, and each independent cavity is correspondingly provided with the nanopore; 6) forming a microchannel structure below the cavity layer, and the microchannel structure is used for injecting an aqueous reaction solution into the independent cavity and injecting an oil-phase liquid seal layer onto the lower surface of the cavity layer; 7) forming an oil-phase liquid seal layer on the lower surface of the cavity layer, and the oil-phase liquid seal layer and the aqueous reaction solution in the independent cavity form an oil-water interface to seal and isolate the aqueous reaction solution in their respective independent cavities.

[0016] Optionally, it further includes the step of preparing an electrode structure, and the electrode structure includes a common electrode disposed in the common liquid cavity and an independent electrode disposed in each independent cavity.

[0017] Optionally, the nanopore described 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 bilayer 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 bilayer includes a phospholipid bilayer.

[0018] 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, and the independent electrode exposes a part of the independent cavity to form a removal window. A common electrode is fabricated in the common liquid cavity, and the melting temperatures of the independent electrode and the common electrode are higher 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; by heating and melting, the conductive metal is removed from the removal window.

[0019] Optionally, the conductive metal includes one of germanium, 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.

[0020] Optionally, the shape of the solid-state nanopore includes one of a cylindrical shape, a conical shape, a tower shape, and a funnel shape.

[0021] As described above, the nanopore detection device, manufacturing method, and application based on the heating seal structure of the present invention have the following beneficial effects:

[0022] The present invention provides a nanopore detection device based on a heating seal structure. After the aqueous solution fills the common liquid cavity and the independent cavities, 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 the independent cavities, and achieving the effect of sealing the independent cavities.

[0023] The present invention forms an air-sealed cavity at the bottom of the independent cavity by heating the bottom of the independent cavity after energizing the heating electrode provided at the bottom of the independent cavity, achieving a good liquid seal effect together with the oil-phase liquid seal layer.

[0024] In the present invention, a conductive metal is formed in the independent cavity, and nanopores corresponding to each independent cavity are simultaneously formed in the barrier layer by applying a breakdown voltage. Then, the conductive metal is removed by heating and melting. On the one hand, the preparation of a nanopore array with high alignment accuracy can be achieved. On the other hand, the preparation cost of the nanopore array can be effectively reduced, and it has the advantages of simple and stable processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 FIG. shows a schematic structural diagram of a nanopore detection device based on a heating sealing structure according to an embodiment of the present invention.

[0026] Figures 2 to 5 FIG. shows a schematic diagram of a nanopore implementation mode of a nanopore detection device based on a heating sealing structure according to an embodiment of the present invention.

[0027] Figure 6 FIG. shows a schematic flow chart of application method steps of a nanopore detection device based on a heating sealing structure according to an embodiment of the present invention.

[0028] Figure 7 FIG. shows a schematic flow chart of manufacturing method steps of a nanopore detection device based on a heating sealing structure according to an embodiment of the present invention.

[0029] DESCRIPTION OF REFERENCE NUMERALS

[0030] 101 Cavity layer

[0031] 102 Independent cavity

[0032] 103 Barrier layer

[0033] 104 Nanopore

[0034] 105 Microchannel structure

[0035] 106 Oil-phase liquid seal layer

[0036] 107 Oil-water interface

[0037] 108 Common liquid cavity

[0038] 109 Independent electrode

[0039] 110 Common electrode

[0040] 111 Heating electrode

[0041] 112 Air-sealed cavity

[0042] Steps S11 - S13

[0043] Steps S21 - S26 DETAILED DESCRIPTION OF THE INVENTION

[0044] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand the 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 embodiments. 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.

[0045] When detailing the embodiments of the present invention, for the convenience of description, 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.

[0046] For the convenience of description, spatial relationship terms such as "beneath", "below", "lower than", "under", "above", "on" may be used herein to describe the relationship between an element or feature shown in the drawings and other elements or features. It will be understood that these spatial relationship terms are intended to encompass other directions of the device in use or operation in addition to 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.

[0047] In the context of the present application, the structure in which the first feature is "above" the second feature described may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features are formed between the first and second features, such that the first and second features may not be in direct contact.

[0048] 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 type, quantity, and ratio of each component in its actual implementation can be arbitrarily changed, and the component layout type may also be more complex.

[0049] As Figures 1 to 5 shown, this embodiment provides a nanopore detection device based on a heating and 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 seal layer 106, and a heating and sealing structure.

[0050] As Figure 1 shown, a plurality of nanopores 104 penetrating through the barrier layer 103 are formed in the barrier layer 103.

[0051] The nanopore 104 includes one of a solid-state nanopore 104 and a biological nanopore 104. The barrier layer 103 of the solid-state nanopore 104 includes an insulating dielectric layer, and the barrier layer 103 of the biological nanopore 104 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 104, and the barrier layer 103 is a silicon nitride layer.

[0052] As Figure 2 shown, in one embodiment, the shape of the solid-state nanopore 104 is cylindrical, and the diameter of the nanopore 104 can be 0.1 to 99 nm. Preferably, the diameter of the nanopore 104 is 1 to 5 nm.

[0053] As Figure 3 shown, in another embodiment, the shape of the solid-state nanopore 104 is conical. The conical nanopore 104 has a minimum pore diameter, which can be 0.1 to 99 nm. Preferably, the minimum pore diameter is 1 to 5 nm. Setting the solid-state nanopore 104 to be conical can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore 104 (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.

[0054] As Figure 4 shown, in yet another embodiment, the shape of the solid-state nanopore 104 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 to 99 nm. Preferably, the minimum pore diameter is 1 to 5 nm. Setting the solid-state nanopore 104 to be tower-shaped can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore 104 (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.

[0055] As Figure 5As shown, in another embodiment, the shape of the solid-state nanopore 104 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 104 to be funnel-shaped can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore 104 (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.

[0056] 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.

[0057] 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 silica, etc. The plurality of independent cavities 102 are etched in the silica by means of photolithography-etching. The independent cavity 102 can be a cylindrical cavity, and the diameter of the cylindrical cavity 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 can also be other shapes such as oval, polygon, etc., and is not limited to the examples listed here.

[0058] 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.

[0059] 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 onto the lower surface of the cavity layer 101.

[0060] 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 squeeze out and replace the aqueous solution in the microchannel and 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, forming an oil-water interface 107 to enclose and isolate 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.

[0061] As Figure 1 shown, the heating and sealing structure includes a heating electrode 111 located at the bottom of the independent cavity 102 for forming an air-sealed cavity 112 at the bottom of the independent cavity 102.

[0062] The heating electrode 111 includes a ring-shaped heating electrode surrounding the bottom of the independent cavity 102. In this embodiment, the heating electrode 111 uses a ring-shaped heating electrode surrounding the bottom of the independent cavity 102, which is beneficial to the formation of the air-sealed cavity 112 and is easy to form an air-sealed cavity 112 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 111 can also use a block electrode, and two or more of the block electrodes are evenly distributed at the bottom of the independent cavity 102 to form an air-sealed cavity 112 at the bottom of the independent cavity 102. It should be noted that the structure of the heating electrode 111 is not limited to the above-listed examples and can be selected according to actual needs.

[0063] As Figure 1 shown, the detection device further includes an electrode structure. The electrode structure includes a common electrode 110 disposed in the common liquid cavity 108 and an independent electrode 109 disposed in each independent cavity 102. The detection device is used for DNA sequence detection. By applying a driving voltage on both sides of the nanopore 104, 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 104. When the DNA strand passes through the nanopore 104, it blocks the ion movement to form 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.

[0064] As Figure 6 shown, the present invention also provides an application method of a nanopore detection device based on a heating and sealing structure, including:

[0065] Step 1) S11, injecting an aqueous reaction solution into the independent cavity 102 based on the microchannel structure 105, where the aqueous reaction solution contains the DNA to be detected and an electrolyte solution, and the electrolyte solution can be, for example, potassium chloride (KCl) solution;

[0066] Step 2) S12, injecting an oil-phase liquid seal layer 106 onto the lower surface of the cavity layer 101 based on the microchannel structure 105. The oil-phase liquid seal layer 106 and the aqueous reaction solution in the independent cavity 102 form an oil-water interface 107 to seal and isolate the aqueous reaction solution in their respective independent cavities 102;

[0067] Step 3) S13, heating through the heating electrode 111 to form an air-sealed cavity 112 at the bottom of the independent cavity, and the air-sealed cavity 112 completely covers the bottom of the independent cavity 102;

[0068] Step 4) S14, applying a driving voltage on both sides of the nanopore 104 to drive the ion movement in the aqueous reaction solution to generate a current, and at the same time driving 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 ion movement to form a blocking current. According to the correspondence between the blocking current and the sequence of the DNA, the sequence of the DNA is determined by measuring the magnitude of the blocking current.

[0069] As Figures 1 to 5 and Figure 7 shown, the present invention also provides a manufacturing method of a nanopore detection device based on a heating and sealing structure, and the manufacturing method includes the steps:

[0070] As Figure 1 and Figure 7 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.

[0071] 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, and the lipid molecule layer includes a phospholipid bilayer. Specifically, according to the different nanopores 104 formed subsequently, the nanopores 104 include one of solid nanopores 104 and biological nanopores 104. The barrier layer 103 of the solid nanopores 104 includes an insulating dielectric layer, and the barrier layer 103 of the biological nanopores 104 includes one of a lipid molecule layer and a block copolymer molecule layer.

[0072] As Figure 1 and Figure 7 shown, then step 2) S22 is performed to etch the substrate to form a common liquid cavity 108.

[0073] As Figure 1 and Figure 7 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.

[0074] The material of the cavity layer 101 can 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 cavity 102 can be a cylindrical cavity, and the diameter of the cylindrical cavity 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 can also be other shapes such as an ellipse or a polygon, and is not limited to the examples listed here.

[0075] 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.

[0076] As Figure 1 and Figure 7 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 111 located at the bottom of the independent cavity 102 for forming an air-sealed cavity 112 at the bottom of the independent cavity 102.

[0077] In this embodiment, the heating electrode 111 includes a ring-shaped heating electrode surrounding the bottom of the independent cavity 102. The heating electrode in this embodiment adopts a ring-shaped heating electrode surrounding the bottom of the independent cavity 102, which is conducive to the formation of the air-sealed cavity 112, and it is easy to form an air-sealed cavity 112 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 111 can also adopt a block electrode, and two or more of the block electrodes are evenly distributed at the bottom of the independent cavity 102 to form an air-sealed cavity 112 at the bottom of the independent cavity 102. It should be noted that the structure of the heating electrode 111 is not limited to the examples listed above and can be selected according to actual needs.

[0078] As Figure 1 and Figure 7 shown, then step 5) S25 is performed to form nanopores 104 in the barrier layer 103, and each independent cavity 102 is correspondingly provided with the nanopores 104.

[0079] The nanopores 104 include one of solid-state nanopores 104 and biological nanopores 104. In this embodiment, the nanopores 104 are solid-state nanopores 104, and the shape of the solid-state nanopores 104 includes one of cylindrical, conical, tower-shaped, and funnel-shaped.

[0080] As Figure 2 shown, in one embodiment, the shape of the solid-state nanopore 104 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.

[0081] As Figure 3 shown, in another embodiment, the shape of the solid-state nanopore 104 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 104 as conical can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore 104 (that is, 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.

[0082] As Figure 4As shown, in another embodiment, the shape of the solid-state nanopore 104 is tower-shaped. The tower-shaped nanopore 104 is composed of two or more circular holes with different diameters connected in sequence. 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 104 to be tower-shaped can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore 104 (i.e., the thickness of the barrier layer 103 corresponding to the minimum pore diameter is smaller), which can 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.

[0083] As Figure 5 shown, in another embodiment, the shape of the solid-state nanopore 104 is funnel-shaped. The funnel-shaped nanopore 104 is composed of two opposite conical holes connected. 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 104 to be funnel-shaped can, on the one hand, effectively reduce the actual thickness of the solid-state nanopore 104 (i.e., the thickness of the barrier layer 103 corresponding to the minimum pore diameter is smaller), which can 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.

[0084] In this embodiment, the method for forming the solid-state nanopore 104 in the barrier layer 103 includes the steps:

[0085] 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 temperature of the independent electrode 109 and the common electrode 110 is greater than the melting temperature of the conductive metal;

[0086] 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;

[0087] Step 5-3): Remove the conductive metal from the removal window by heating and melting.

[0088] For example, the conductive metal includes one of germanium, 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.

[0089] 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, the preparation of a nanopore 104 array with high alignment accuracy can be achieved. On the other hand, the preparation cost of the nanopore 104 array can be effectively reduced, and it has the advantages of simple and stable processes.

[0090] As Figure 1 and Figure 7 shown, then step 6) S26 is carried out to form a microchannel structure 105 under the cavity layer 101. The microchannel structure 105 is used to inject an aqueous reaction solution into the independent cavity 102 and inject an oil-phase liquid seal layer 106 onto the lower surface of the cavity layer 101.

[0091] As Figure 1 and Figure 7 shown, then step 7) S27 is carried out to form an oil-phase liquid seal layer 106 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 seal and isolate the aqueous reaction solution in their respective independent cavities 102.

[0092] 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 sealed in the independent cavity 102 by the oil-phase liquid seal layer 106 to form an oil-water interface 107, sealing 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.

[0093] As described above, the nanopore detection device, manufacturing method, and application based on the heating seal structure of the present invention have the following beneficial effects:

[0094] The present invention provides a nanopore detection device based on a heating and sealing structure. After the aqueous solution fills the common liquid chamber and the independent chambers, an oil-phase liquid sealing layer is injected through a microchannel. The oil-phase liquid sealing 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 sealing layer, but will be sealed in the independent chamber by the oil-phase liquid sealing 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.

[0095] The present invention sets a heating electrode at the bottom of the independent chamber. After being powered on, the bottom of the independent chamber is heated to form an air-sealed chamber at the bottom of the independent chamber, jointly achieving a good liquid-sealing effect with the oil-phase liquid sealing layer.

[0096] The present invention forms a conductive metal in the independent chamber, and forms nanopores corresponding to each independent chamber 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 achieve the preparation of a nanopore array with high alignment accuracy. On the other hand, it can effectively reduce the preparation cost of the nanopore array, having the advantages of simple and stable processes.

[0097] Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0098] 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 ideas 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 heating and sealing structure, characterized in that, 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; the nanopores include one of solid-state nanopores and biological nanopores; 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 seal layer onto the lower surface of the cavity layer; an oil-phase liquid seal layer, located on the lower surface of the cavity layer, and the oil-phase liquid seal 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 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; further including an electrode structure, the electrode structure including 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 heating and sealing structure according to claim 1, characterized in that: the barrier layer of the solid-state nanopores includes an insulating dielectric layer, and the barrier layer of the biological nanopores 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.

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

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

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

6. The nanopore detection device based on a heating and 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 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.

8. The nanopore detection device based on 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 across the two 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 corresponding relationship between the blocking current and the DNA sequence, the DNA sequence is determined by measuring the magnitude of the blocking current.

9. A method for applying a nanopore detection device based on a heating and sealing structure according to any one of claims 1 to 8, characterized in that, comprising: 1) Injecting an aqueous reaction solution into the independent cavity based on the microchannel structure; 2) Injecting an oil-phase liquid sealing layer onto the lower surface of the cavity layer based on the microchannel structure. The oil-phase liquid sealing layer forms an oil-water interface with the aqueous reaction solution in the independent cavity 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 across the two 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 corresponding relationship between the blocking current and the DNA sequence, the DNA sequence is determined by measuring the magnitude of the blocking current.

10. A method for manufacturing a nanopore detection device based on a heating and sealing structure, characterized in that, 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 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) Forming a nanopore in the barrier layer, and each independent cavity is correspondingly provided with the nanopore; 6) Forming a microchannel structure below the cavity layer. The microchannel structure is 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 on the lower surface of the cavity layer. The oil-phase liquid sealing layer forms an oil-water interface with the aqueous reaction solution in the independent cavity to seal and isolate the aqueous reaction solution in their respective independent cavities; It further includes the step of preparing an electrode structure. The electrode structure includes a common electrode disposed in the common liquid cavity and an independent electrode disposed in each independent cavity.

11. According to the method for manufacturing a nanopore detection device based on a heating and sealing structure according to claim 10, characterized in that: The nanopore described in step 5) 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.

12. The manufacturing method of the nanopore detection device based on the heating and sealing structure according to claim 10, characterized in that: The nanopore described in step 5) 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.

13. The manufacturing method of the nanopore detection device based on the heating and sealing structure according to claim 12, characterized in that: 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, manufacturing a common electrode in the common liquid cavity, and the melting temperature of the independent electrode and the common electrode being greater 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.

14. The manufacturing method of the nanopore detection device based on the heating and sealing structure according to claim 13, characterized in that: The conductive metal includes one of germanium, 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.

15. The manufacturing method of the nanopore detection device based on the heating and sealing structure according to claim 13, characterized in that: The shape of the solid-state nanopore includes one of cylindrical, conical, tower-shaped, and funnel-shaped.

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