CN115125133A - Nanopore detection device, fabrication method and application based on heating and sealing structure - Google Patents

Abstract

The invention provides a nanopore detection device based on a heating and sealing structure, a manufacturing method and an application. The device includes: a barrier layer with a plurality of nanopores and a common liquid cavity above; a cavity layer, including a plurality of independent cavities, each Each independent cavity is correspondingly equipped with nanopores; the micro-channel structure is used to inject the solution; the oil-phase liquid sealing layer, the oil-phase liquid sealing layer and the water-phase reaction solution in the independent cavity form an oil-water interface, so that the water-phase liquid sealing layer forms an oil-water interface. The reaction solution is closed and isolated in the respective independent chambers; the heating and sealing structure is used to form an air-enclosed chamber at the bottom of the independent chambers. The invention seals and isolates the water-phase reaction solution in the respective independent cavities through the oil-phase liquid sealing layer, prevents the possible cross-leakage of the salt solution between the independent cavities, and realizes the effect of sealing the independent cavities. The electrode forms an air-enclosed cavity at the bottom of the independent cavity, which together with the oil-phase liquid-sealing layer achieves a good liquid-sealing effect.

Description

Nanopore detection device, fabrication method and application based on heating and sealing structure

technical field

The invention belongs to the field of biological detection devices and manufacturing, and in particular relates to a nanopore detection device based on a heating and sealing structure, a manufacturing method and an application.

Background technique

Most of the current nanopore sequencing technologies use the form of measuring ionic current to measure the blocking current generated by DNA vias. According to the difference in size information and charge information of different bases, the size of the blocking current is different, so different bases correspond to different The blocking current can be used to analyze the sequence information of DNA. Nanopores are generally on an insulating film, for example, biological nanopores are embedded in an insulating lipid bilayer film. Solid-state nanopores are prepared in solid state by semiconductor processing technology on the insulating film; the nanopore and insulating film are placed in a dielectric solution (usually KCl solution) to separate the solution into two parts; a driving voltage is applied on both sides of the insulating film, and this driving voltage has two functions: on the one hand, the voltage It will drive the charged ions in the salt solution to pass through the nanopore, and the movement of the charged ions generates an ionic current through the pore; the other effect of the driving voltage is to drive the charged DNA molecules to move through the nanopore, and the DNA molecules move in the nanopore. When the movement of ions in the nanopore is blocked, the intensity of the ionic current will decrease, forming a blocking current; because the size and charge information of the four bases of DNA are different, the blocking current generated by different bases is different, which It is the basic principle of nanopore sequencing.

If the throughput of sequencing needs to be improved, a large number of nanopores need to be sequenced at the same time. Nanopores are often prepared on nanopore array chips. The nanopore array chips can share a solution system and a common electrode, but each nanopore also needs There are independent electrodes and independent solution chambers, and there needs to be sufficient sealing conditions between these independent electrodes and solution chambers, and the salt solution between the independent chambers cannot leak, otherwise the ions generated in each nanopore The current signal will have leakage current and crosstalk, which will cause unfavorable phenomena such as signal noise increase and cross-interference, which will affect the accurate interpretation of the signal.

SUMMARY OF THE INVENTION

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a nanopore detection device based on a heat-sealed structure, a manufacturing method and an application, which are used to solve the problem of generation in each nanopore of the nanopore array in the prior art. The ionic current signal is prone to leakage current and crosstalk problems.

In order to achieve the above object and other related objects, the present invention provides a nanopore detection device based on a heat-sealed structure, the detection device includes: a barrier layer, wherein a plurality of nanopores penetrating the barrier layer are formed in the barrier layer , there is a common liquid cavity above the barrier layer; the cavity layer, located below the barrier layer, includes a plurality of independent cavities, each of which is correspondingly configured with the nanopores; the micro-channel structure is located in below the cavity layer, for injecting the aqueous reaction solution into the independent cavity, and injecting the oil-phase liquid sealing layer to the lower surface of the cavity layer; the oil-phase liquid sealing layer is located in the cavity The lower surface of the layer, the oil-phase liquid sealing layer and the water-phase reaction solution in the independent cavity form an oil-water interface, so as to seal and isolate the water-phase reaction solution in the independent cavity; the heating sealing structure, A heating electrode located at the bottom of the independent cavity is included to form an air-enclosed cavity at the bottom of the independent cavity.

Optionally, the nanopore includes one of a solid nanopore and a biological nanopore, the barrier layer of the solid nanopore includes an insulating medium layer, and the barrier layer of the biological nanopore includes a lipid molecule layer and One of the block copolymer molecular layers, 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. The lipid molecule layer comprises a phospholipid bilayer.

Optionally, the shape of the solid nanopore includes one of cylindrical shape, cone shape, tower shape and funnel shape.

Optionally, the minimum pore size of the nanopore is 0.1-99 nm.

Optionally, a plurality of the nanopores in the barrier layer and a plurality of the independent cavities in the cavity layer are arranged in a periodic array.

Optionally, an electrode structure is also included, and the electrode structure includes a common electrode disposed in the common liquid cavity and an independent electrode disposed in each of the independent cavities.

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.

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

Optionally, the detection device is used for the detection of DNA sequences, and a driving voltage is applied on both sides of the nanopore to drive the movement of ions in the aqueous reaction solution to generate current, and at the same time drive the DNA chain to pass through the Nanopore, the DNA chain blocks the movement of the ions when passing through the nanopore to form a blocking current, and the magnitude of the blocking current is determined according to the corresponding relationship between the blocking current and the DNA sequence to determine the sequence of the DNA.

The present invention also provides an application method of a nanopore detection device based on a heating and sealing structure, comprising: 1) injecting an aqueous reaction solution into the independent cavity based on the microfluidic structure; 2) based on the microfluidic structure The channel structure injects the oil-phase liquid sealing layer into the lower surface of the cavity layer, and the oil-phase liquid sealing layer forms an oil-water interface with the water-phase reaction solution in the independent cavity to seal the water-phase reaction solution. and isolated in their respective independent cavities; 3) heating by the heating electrodes to form an air-enclosed cavity at the bottom of the independent cavities; 4) by applying a driving voltage on both sides of the nanopores to drive the The movement of ions in the aqueous reaction solution generates an electric current, and at the same time drives the DNA strands in the aqueous reaction solution to pass through the nanopore, and the DNA strands block the movement of the ions when passing through the nanopore. , 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 blocking current.

The present invention also provides a method for fabricating a nanopore detection device based on a heating and sealing structure, the fabrication method includes steps: 1) providing a substrate, forming a dielectric layer on the substrate, and forming a barrier 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) in the A heat-sealing structure is formed at the bottom of the independent cavity, and the heat-sealing structure includes a heating electrode located at the bottom of the independent cavity for forming an air-enclosed cavity at the bottom of the independent cavity; 5) forming nanometers in the barrier layer 6) A micro-channel structure is formed under the cavity layer, and the micro-channel structure is used for injecting the aqueous reaction solution into the independent cavity cavity, and injecting an oil-phase liquid sealing layer into 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 and the independent cavity The water-phase reaction solution in the water-phase reaction solution forms an oil-water interface, so as to seal and isolate the water-phase reaction solution in the respective independent cavities.

Optionally, the method further includes the step of preparing an electrode structure, wherein the electrode structure includes a common electrode disposed in the common liquid cavity and an independent electrode disposed in each of the independent cavities.

Optionally, in step 5) the nanopores include one of solid nanopores and biological nanopores, the barrier layer of the solid nanopores includes an insulating medium layer, and the barrier layer of the biological nanopores includes lipids. One of molecular layer and block copolymer molecular layer, the insulating dielectric layer includes silicon nitride, silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide and graphene In one, the lipid molecular layer comprises a phospholipid bilayer.

Optionally, the method for forming solid nanopores in the barrier layer includes the steps of: forming a conductive metal on the independent cavity; forming an independent electrode corresponding to each of the independent cavities on the conductive metal, so that the The independent electrode exposes a part of the independent cavity to form a removal window, and a common electrode is fabricated in the common liquid cavity, and the melting temperature of the independent electrode and the common electrode is higher than the melting temperature of the conductive metal; The independent electrode and the common electrode apply a breakdown voltage, so that the conductive metal breaks down the barrier layer, so as to simultaneously form nanopores corresponding to each independent cavity in the barrier layer; The removal window removes the conductive metal.

Optionally, the conductive metal includes one of germanium, tin, indium and bismuth, and the material of the independent electrode and the common electrode includes one of copper, aluminum, titanium nitride, gold and platinum.

Optionally, the shape of the solid nanopore includes one of cylindrical shape, cone shape, tower shape and funnel shape.

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

The invention provides a nanopore detection device based on a heating and sealing structure. After the water phase solution fills the common liquid cavity and the independent cavity, it is injected into the oil phase liquid sealing layer through the microfluidic channel. The water phase solution in the channel is squeezed out and replaced, covering the lower surface of the cavity layer. Under the action of surface tension, the water phase solution in the independent cavity will not be replaced by the oil phase liquid seal layer, but by the oil phase liquid seal. The layer is enclosed in the independent cavity to form an oil-water interface, and the aqueous reaction solution is sealed and isolated in the independent cavity to prevent the cross leakage of salt solution that may occur between the independent cavities and achieve the effect of sealing the independent cavity. .

In the present invention, a heating electrode is arranged at the bottom of the independent cavity, and the bottom of the independent cavity is heated after power-on, so as to form an air-sealed cavity at the bottom of the independent cavity, and a good liquid sealing effect is achieved together with the oil-phase liquid sealing layer. .

In the present invention, 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, and then the conductive metal is removed by heating and melting. The preparation of nanohole arrays with high alignment precision can be realized, and on the other hand, the preparation cost of nanohole arrays can be effectively reduced, and the process is simple and stable.

Description of drawings

FIG. 1 is a schematic structural diagram of a nanopore detection device based on a heat-sealed structure according to an embodiment of the present invention.

2 to 5 are schematic diagrams illustrating nanopore implementations of a nanopore detection device based on a heat-sealed structure according to an embodiment of the present invention.

FIG. 6 is a schematic flow chart showing the steps of the application method of the nanopore detection device based on the heating and sealing structure according to the embodiment of the present invention.

FIG. 7 is a schematic flowchart showing the steps of a manufacturing method of a nanopore detection device based on a heat-sealed structure according to an embodiment of the present invention.

Component label description

101 Cavity layer

102 independent chambers

103 Barrier layer

104 nanopore

105 Microfluidic Structure

106 Oil phase liquid seal

107 Oil-water interface

108 Common liquid chamber

109 individual electrodes

110 Common electrode

111 Heating electrode

112 Air-enclosed chamber

Steps S11～S13

Steps S21～S26

Detailed ways

The embodiments of the present invention are described below through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and 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.

When describing the embodiments of the present invention in detail, for the convenience of explanation, the cross-sectional views showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the protection scope of the present invention. In addition, the three-dimensional spatial dimensions of length, width and depth should be included in the actual production.

For convenience of description, spatially relative terms such as "below," "below," "below," "below," "above," "on," etc. may be used herein to describe an element shown in the figures or The relationship of a feature to other components or features. It will be understood that these spatially relative terms are intended to encompass other directions of the device in use or operation than those depicted in the figures. In addition, when a layer is referred to as being 'between' two layers, it can be the only layer between the two layers, or one or more intervening layers may also be present.

In the context of this application, descriptions of structures where a first feature is "on" a second feature can include embodiments in which the first and second features are formed in direct contact, and can also include further features formed over the first and second features. Embodiments between the second features such that the first and second features may not be in direct contact.

It should be noted that the diagrams provided in this embodiment are only to illustrate the basic concept of the present invention in a schematic way, so the diagrams only show the components related to the present invention rather than the number, shape and the number of components in the actual implementation. For dimension drawing, the type, quantity and proportion of each component can be changed at will in actual implementation, and the component layout may also be more complicated.

As shown in FIGS. 1 to 5 , this embodiment provides a nanopore detection device based on a heat-sealed structure. The detection device includes: a common liquid cavity 108 , a barrier layer 103 , a cavity layer 101 , and a micro-channel structure 105 , the oil phase liquid sealing layer 106 and the heating sealing structure.

As shown in FIG. 1 , the blocking layer 103 is formed with a plurality of nanopores 104 penetrating the blocking layer 103 .

The nanopore 104 includes one of a solid nanopore 104 and a biological nanopore 104 , the barrier layer 103 of the solid nanopore 104 includes an insulating medium layer, and the barrier layer 103 of the biological nanopore 104 includes lipid One of molecular layer and block copolymer molecular layer, the insulating dielectric layer includes silicon nitride, silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide and graphene In one, the lipid molecular layer comprises a phospholipid bilayer. In this embodiment, the nanoholes 104 are solid nanoholes 104 , and the blocking layer 103 is a silicon nitride layer.

As shown in FIG. 2 , in one embodiment, the solid nanopore 104 is cylindrical in shape, and the diameter of the nanopore 104 may be 0.1˜99 nm. Preferably, the diameter of the nanopore 104 is 1˜99 nm. 5nm.

As shown in FIG. 3 , in another embodiment, the shape of the solid nanopore 104 is tapered, and the tapered nanopore 104 has a minimum pore size, and the minimum pore size may be 0.1-99 nm. The minimum pore size is 1 to 5 nm. Setting the solid-state nanopore 104 into a tapered shape can effectively reduce the actual thickness of the solid-state nanopore 104 (that is, the thickness of the barrier layer 103 corresponding to the smallest aperture is smaller), and can ensure the measurement accuracy while ensuring the measurement accuracy. The nanopore 104 is prevented from being completely blocked, and the flow of DNA in the aqueous reaction solution is ensured. On the other hand, the actual moving distance of the DNA in the nanopore 104 can be reduced, and the detection accuracy can be improved.

As shown in FIG. 4 , in another embodiment, the solid nanopore 104 is in the shape of a tower, and the tower-shaped nanopore 104 includes two or more circular holes with different diameters connected in sequence. The shaped nanopore 104 has a minimum pore size, and the minimum pore size can be 0.1-99 nm, preferably, the minimum pore size is 1-5 nm. Setting the solid-state nanopore 104 in a tower shape can effectively reduce the actual thickness of the solid-state nanopore 104 (that is, the thickness of the barrier layer 103 corresponding to the minimum aperture is small), and can ensure the measurement accuracy while ensuring the measurement accuracy. The nanopore 104 is prevented from being completely blocked, and the flow of DNA in the aqueous reaction solution is ensured. On the other hand, the actual moving distance of the DNA in the nanopore 104 can be reduced, and the detection accuracy can be improved.

As shown in FIG. 5 , in another embodiment, the shape of the solid nanopore 104 is a funnel shape, and the funnel-shaped nanopore 104 includes two opposite conical pores connected. 104 has a minimum pore size, and the minimum pore size can be 0.1-99 nm, preferably, the minimum pore size is 1-5 nm. Setting the solid nanopore 104 into a funnel shape, on the one hand, can effectively reduce the actual thickness of the solid nanopore 104 (that is, the thickness of the barrier layer 103 corresponding to the minimum aperture is small), while ensuring the measurement accuracy, The nanopore 104 is prevented from being completely blocked, and the flow of DNA in the aqueous reaction solution is ensured. On the other hand, the actual moving distance of the DNA in the nanopore 104 can be reduced, and the detection accuracy can be improved.

As shown in FIG. 1 , the common liquid cavity 108 is located above the barrier layer 103 and is used to carry an aqueous reaction solution. The aqueous reaction solution in the common liquid cavity 108 can be injected directly or injected through a micro-channel structure .

As shown in FIG. 1 , the cavity layer 101 is located under the barrier layer 103 , the cavity layer 101 includes a plurality of independent cavities 102 , and each of the independent cavities 102 is correspondingly configured with one of the nanopores 104. The material of the cavity layer 101 can be silicon dioxide or the like, and the plurality of independent cavities 102 are etched from the silicon dioxide by means of photolithography-etching, and the independent cavities 102 can be cylindrical hollow. 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 may also be other shapes such as ellipse, polygon, etc., which is not limited to the examples listed here.

In this embodiment, the plurality of nanoholes 104 in the blocking layer 103 and the plurality of independent cavities 102 in the cavity layer 101 are arranged in a periodic array, so as to improve the detection throughput. quantity and efficiency.

As shown in FIG. 1 , the microfluidic channel structure 105 is located under the cavity layer 101 , and is used for injecting the aqueous reaction solution into the independent cavity 102 and injecting the oil-phase liquid sealing layer 106 into the independent cavity 102 . The lower surface of the cavity layer 101 .

As shown in FIG. 1 , the oil-phase liquid sealing layer 106 is located on the lower surface of the cavity layer 101 , and the oil-phase liquid sealing layer 106 forms an oil-water interface 107 with the water-phase reaction solution in the independent cavity 102 , So as to seal and isolate the aqueous reaction solution in the respective independent chambers 102 . After the water-phase solution fills the common liquid cavity 108 and the independent cavity 102, it is injected into the oil-phase liquid sealing layer 106 through the micro-channel, and the oil-phase liquid-sealing layer 106 squeezes and replaces the water-phase solution in the micro-channel, covering The lower surface of the cavity layer 101, as shown in FIG. 1, under the action of surface tension, the water phase solution in the independent cavity 102 will not be replaced by the oil phase liquid sealing layer 106, but is sealed by the oil phase liquid. The layer 106 is enclosed in the independent cavity 102 to form an oil-water interface 107, and the water-phase reaction solution is closed and isolated in the independent cavity 102 to prevent the cross leakage of salt solution that may occur between the independent cavities 102, and to achieve independent The effect of the cavity 102 being closed.

As shown in FIG. 1 , 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 .

The heating electrode 111 includes a ring-shaped heating electrode surrounding the bottom of the independent cavity 102 . The heating electrode 111 in this embodiment adopts a ring-shaped heating electrode surrounding the bottom of the independent cavity 102 , which is beneficial to the air-enclosed cavity 112 . It is easy to form an air-enclosed cavity 112 that completely covers the entire bottom of the independent cavity 102 , which can effectively realize the sealing and isolation of the independent cavity 102 . Of course, in other embodiments, the heating electrode 111 may also be a block electrode, and two or more of the block electrodes are evenly distributed at the bottom of the independent cavity 102 , so that the bottom of the independent cavity 102 To form the air-enclosed cavity 112, 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.

As shown in FIG. 1 , the detection device further includes an electrode structure including a common electrode 110 disposed in the common liquid chamber 108 and an independent electrode 109 disposed in each of the independent chambers 102 . The detection device is used for the detection of DNA sequences, by applying a driving voltage on both sides of the nanopore 104 to drive the movement of ions in the aqueous reaction solution to generate current, and at the same time drive the DNA chain to pass through the nanopore 104 , the DNA chain blocks the movement of the ions when passing through the nanopore 104 to form a blocking current. According to the corresponding relationship between the blocking current and the DNA sequence, the magnitude of the blocking current is determined to determine The DNA sequence was determined.

As shown in FIG. 6 , the present invention also provides an application method of the nanopore detection device based on the heating and sealing structure, including:

Step 1) S11, injecting an aqueous reaction solution into the independent cavity 102 based on the microfluidic channel structure 105, the aqueous reaction solution containing the DNA to be tested and a dielectric solution, the dielectric solution, for example, can be chlorinated Potassium (KCl) solution;

Step 2) S12, injecting the oil-phase liquid sealing layer 106 into the lower surface of the cavity layer 101 based on the micro-channel structure 105, the oil-phase liquid-sealing layer 106 and the water phase in the independent cavity 102 The reaction solution forms an oil-water interface 107 to seal and isolate the water-phase reaction solution in the respective independent chambers 102;

Step 3) S13, heating by the heating electrode 111 to form an air-enclosed cavity 112 at the bottom of the independent cavity, the air-enclosed cavity 112 completely covering the bottom of the independent cavity 102;

Step 4) S14, by applying a driving voltage on both sides of the nanopore 104, to drive the movement of ions in the aqueous reaction solution to generate current, and at the same time drive the DNA strands in the aqueous reaction solution to pass through the nanometer. Pore 104, when the DNA strand passes through the nanopore 104, the movement of the ions is blocked to form a blocking current. size to determine the sequence of the DNA.

As shown in FIG. 1 to FIG. 5 and FIG. 7 , the present invention also provides a manufacturing method of a nanopore detection device based on a heat-sealed structure. The manufacturing method includes the steps:

As shown in FIG. 1 and FIG. 7 , step 1) S21 is first performed, a substrate is provided, a dielectric layer is formed on the substrate, and a barrier layer 103 is formed on the dielectric layer.

In this embodiment, the substrate is a silicon substrate, the dielectric layer is a silicon dioxide layer, the blocking layer 103 includes an insulating medium layer or a lipid molecule layer, and the insulating medium layer includes silicon nitride, One of silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide and graphene, the lipid molecular layer includes a phospholipid bilayer, specifically, according to the subsequent formation Different from the nanopore 104, the nanopore 104 includes one of a solid nanopore 104 and a biological nanopore 104, the barrier layer 103 of the solid nanopore 104 includes an insulating medium layer, and all the biological nanopores 104 The blocking layer 103 includes one of a lipid molecular layer and a block copolymer molecular layer.

As shown in FIG. 1 and FIG. 7 , step 2) S22 is performed, and the substrate is etched to form the common liquid cavity 108 .

As shown in FIG. 1 and FIG. 7 , step 3) S23 is performed to etch the dielectric layer to form a plurality of independent cavities 102 in the dielectric layer to form the cavity layer 101 .

The material of the cavity layer 101 can be silicon dioxide or the like, and the plurality of independent cavities 102 are etched from the silicon dioxide by means of photolithography-etching, and the independent cavities 102 can be cylindrical hollow. 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 may also be other shapes such as ellipse, polygon, etc., which is not limited to the examples listed here.

In this embodiment, the plurality of nanoholes 104 in the blocking layer 103 and the plurality of independent cavities 102 in the cavity layer 101 are arranged in a periodic array, so as to improve the detection throughput. quantity and efficiency.

As shown in FIG. 1 and FIG. 7 , then step 4) S24 is performed to form a heating sealing structure at the bottom of the independent cavity 102 , and the heating sealing structure includes a heating electrode 111 located at the bottom of the independent cavity 102 for An air-enclosed cavity 112 is formed at the bottom of the independent cavity 102 .

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 can be beneficial to The formation of the air-enclosed cavity 112 can easily form an air-enclosed cavity 112 that completely covers the entire bottom of the independent cavity 102 , which can effectively realize the sealing and isolation of the independent cavity 102 . Of course, in other embodiments, the heating electrode 111 may also be a block electrode, and two or more of the block electrodes are evenly distributed at the bottom of the independent cavity 102 , so that the bottom of the independent cavity 102 To form the air-enclosed cavity 112, 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.

As shown in FIG. 1 and FIG. 7 , step 5) S25 is performed to form nanoholes 104 in the barrier layer 103 , and each of the independent cavities 102 is correspondingly configured with the nanoholes 104 .

The nanopore 104 includes one of a solid nanopore 104 and a biological nanopore 104. In this embodiment, the nanopore 104 is a solid nanopore 104, and the shape of the solid nanopore 104 includes a cylinder, a cone, and a One of the shape, tower shape and funnel shape.

As shown in FIG. 2 , in one embodiment, the solid nanopore 104 is cylindrical in shape, and the diameter of the nanopore 104 may be 0.1˜99 nm. Preferably, the diameter of the nanopore 104 is 1˜99 nm. 5nm.

As shown in FIG. 3 , in another embodiment, the shape of the solid nanopore 104 is tapered, and the tapered nanopore 104 has a minimum pore size, and the minimum pore size may be 0.1-99 nm. The minimum pore size is 1 to 5 nm. Setting the solid-state nanopore 104 into a tapered shape can effectively reduce the actual thickness of the solid-state nanopore 104 (that is, the thickness of the barrier layer 103 corresponding to the smallest aperture is smaller), and can ensure the measurement accuracy while ensuring the measurement accuracy. The nanopore 104 is prevented from being completely blocked, and the flow of DNA in the aqueous reaction solution is ensured. On the other hand, the actual moving distance of the DNA in the nanopore 104 can be reduced, and the detection accuracy can be improved.

As shown in FIG. 4 , in another embodiment, the solid nanopore 104 is in the shape of a tower, and the tower-shaped nanopore 104 includes two or more circular holes with different diameters connected in sequence. The shaped nanopore 104 has a minimum pore size, and the minimum pore size can be 0.1-99 nm, preferably, the minimum pore size is 1-5 nm. Setting the solid-state nanopore 104 in a tower shape can effectively reduce the actual thickness of the solid-state nanopore 104 (that is, the thickness of the barrier layer 103 corresponding to the minimum aperture is small), and can ensure the measurement accuracy while ensuring the measurement accuracy. The nanopore 104 is prevented from being completely blocked, and the flow of DNA in the aqueous reaction solution is ensured. On the other hand, the actual moving distance of the DNA in the nanopore 104 can be reduced, and the detection accuracy can be improved.

As shown in FIG. 5 , in another embodiment, the shape of the solid nanopore 104 is a funnel shape, and the funnel-shaped nanopore 104 includes two opposite conical pores connected. 104 has a minimum pore size, and the minimum pore size can be 0.1-99 nm, preferably, the minimum pore size is 1-5 nm. Setting the solid nanopore 104 into a funnel shape, on the one hand, can effectively reduce the actual thickness of the solid nanopore 104 (that is, the thickness of the barrier layer 103 corresponding to the minimum aperture is small), while ensuring the measurement accuracy, The nanopore 104 is prevented from being completely blocked, and the flow of DNA in the aqueous reaction solution is ensured. On the other hand, the actual moving distance of the DNA in the nanopore 104 can be reduced, and the detection accuracy can be improved.

In this embodiment, the method for forming the solid nanopores 104 in the blocking layer 103 includes the steps of:

Step 5-1) forming a conductive metal on the independent cavity 102; forming an independent electrode 109 corresponding to each independent cavity 102 on the conductive metal, and the independent electrode 109 exposes part of the independent cavity 102 to form a removal window, a common electrode 110 is fabricated in the common liquid chamber 108, and the melting temperature of the independent electrode 109 and the common electrode 110 is greater than the melting temperature of the conductive metal;

Step 5-2) By applying a breakdown voltage to the independent electrode 109 and the common electrode, the conductive metal breaks down the barrier layer 103, so as to simultaneously form and each independent cavity 102 in the barrier layer 103 the corresponding nanopore 104;

Step 5-3) The conductive metal is removed from the removal window by heating and melting.

For example, the conductive metal includes one of germanium, tin, indium, and bismuth, and the material of the independent electrode 109 and the common electrode 110 includes one of copper, aluminum, titanium nitride, gold, and platinum.

In the present invention, 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, and then the conductive metal is removed by heating and melting. On the one hand, the preparation of the nanohole 104 array with high alignment precision can be realized, and on the other hand, the preparation cost of the nanohole 104 array can be effectively reduced, and the process is simple and stable.

As shown in FIG. 1 and FIG. 7 , then step 6) S26 is performed, and a micro-channel structure 105 is formed under the cavity layer 101 , and the micro-channel structure 105 is used for injecting the aqueous reaction solution into the independent cavity 102 , and injecting the oil-phase liquid sealing layer 106 into the lower surface of the cavity layer 101 .

As shown in FIG. 1 and FIG. 7 , then step 7) S27 is performed to form an oil-phase liquid sealing layer 106 on the lower surface of the cavity layer 101 . The water-phase reaction solution forms an oil-water interface 107 to seal and isolate the water-phase reaction solution in the respective independent cavities 102 .

As shown in FIG. 1 , under the action of surface tension, the water-phase solution in the independent cavity 102 will not be replaced by the oil-phase liquid sealing layer 106 , but is enclosed in the independent cavity 102 by the oil-phase liquid sealing layer 106 . The oil-water interface 107 is formed to seal and isolate the water-phase reaction solution in the respective independent chambers 102 , so as to prevent the possible cross leakage of salt solution between the independent chambers 102 and achieve the effect of sealing the independent chambers 102 .

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

The invention provides a nanopore detection device based on a heating and sealing structure. After the water phase solution fills the common liquid cavity and the independent cavity, it is injected into the oil phase liquid sealing layer through the microfluidic channel. The water phase solution in the channel is squeezed out and replaced, covering the lower surface of the cavity layer. Under the action of surface tension, the water phase solution in the independent cavity will not be replaced by the oil phase liquid seal layer, but by the oil phase liquid seal. The layer is enclosed in the independent cavity to form an oil-water interface, and the aqueous reaction solution is sealed and isolated in the independent cavity to prevent the cross leakage of salt solution that may occur between the independent cavities and achieve the effect of sealing the independent cavity. .

In the present invention, a heating electrode is arranged at the bottom of the independent cavity, and the bottom of the independent cavity is heated after power-on, so as to form an air-sealed cavity at the bottom of the independent cavity, and a good liquid sealing effect is achieved together with the oil-phase liquid sealing layer. .

In the present invention, 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, and then the conductive metal is removed by heating and melting. The preparation of nanohole arrays with high alignment precision can be realized, and on the other hand, the preparation cost of nanohole arrays can be effectively reduced, and the process is simple and stable.

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

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims (16)

1. A nanopore detection device based on a heated sealing structure, the detection device comprising:

a barrier layer having a plurality of nanopores formed therein that extend through the barrier layer, a common liquid chamber above the barrier layer;

the cavity layer is positioned below the blocking layer and comprises a plurality of independent cavities, and each independent cavity is correspondingly provided with the nanopore;

the micro-channel structure is positioned below the cavity layer and used for injecting a water-phase reaction solution into the independent cavity and injecting an oil-phase liquid seal layer into the lower surface of the cavity layer;

the oil phase liquid seal layer is positioned on the lower surface of the cavity layer and forms an oil-water interface with the water phase reaction solution in the independent cavity so as to seal and isolate the water phase reaction solution in the independent cavity;

and the heating sealing structure comprises a heating electrode positioned at the bottom of the independent cavity and is used for forming an air sealing cavity at the bottom of the independent cavity.

2. The nanopore sensing device based on a heated sealing structure of claim 1, wherein: the nanopore comprises one of a solid nanopore and a biological nanopore, a blocking layer of the solid nanopore comprises an insulating medium layer, the blocking layer of the biological nanopore comprises one of a lipid molecule layer and a block copolymer molecule layer, the insulating medium layer comprises 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 comprises a phospholipid bilayer.

3. The nanopore sensing device based on a heated sealing structure of claim 2, wherein: the solid state nanopore has a shape including one of a cylinder, a cone, a tower, and a funnel.

4. The nanopore sensing device based on a heated sealing structure of claim 1, wherein: the minimum aperture of the nanopore is 0.1-99 nm.

5. The nanopore sensing device based on a heated sealing structure of claim 1, wherein: a plurality of the nanopores in the barrier layer and a plurality of the independent cavities in the cavity layer are all arranged in a periodic array.

6. The nanopore sensing device based on a heated sealing structure of claim 1, wherein: the electrode structure comprises a common electrode arranged in the common liquid cavity and independent electrodes arranged in each independent cavity.

7. The nanopore sensing device based on a heated sealing structure of claim 1, wherein: the independent cavity is a cylindrical cavity, the diameter of the cylindrical cavity is 1-1000 mu m, and the interval between two adjacent cylindrical cavities is 2-5000 mu m.

8. The nanopore detection device based on the molecular sealing layer and the heating sealing structure of claim 1, wherein: the heating electrode comprises an annular heating electrode which surrounds the bottom of the independent cavity.

9. The nanopore sensing device based on a heated sealing structure of claim 1, wherein: the detection device is used for detecting a DNA sequence, a driving voltage is applied to two sides of the nanopore to drive ions in the aqueous phase reaction solution to move to generate current, a DNA chain is driven to pass through the nanopore at the same time, the DNA chain blocks the ions when passing through the nanopore to form a blocking current, and the sequence of the DNA is determined by measuring the magnitude of the blocking current according to the corresponding relation between the blocking current and the sequence of the DNA.

10. The application method of the nanopore detection device based on the heating sealing structure as claimed in any one of claims 1 to 9, comprising:

1) injecting an aqueous phase reaction solution into the independent cavity based on the micro flow channel structure;

2) injecting an oil phase liquid seal layer to the lower surface of the cavity layer based on the micro-channel structure, wherein the oil phase liquid seal layer and the water phase reaction solution in the independent cavity form an oil-water interface so as to seal and isolate the water phase reaction solution in the independent cavity;

3) heating through the heating electrode to form an air closed cavity at the bottom of the independent cavity;

4) and applying a driving voltage on two sides of the nanopore to drive ions in the aqueous phase reaction solution to move to generate current, simultaneously driving a DNA chain in the aqueous phase reaction solution to pass through the nanopore, blocking the ions when the DNA chain passes through the nanopore to form a blocking current, and determining the sequence of the DNA by measuring the magnitude of the blocking current according to the corresponding relation between the blocking current and the sequence of the DNA.

11. A manufacturing method of a nanopore detection device based on a heating sealing structure is characterized by comprising the following 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, wherein the heating and sealing structure comprises a heating electrode positioned at the bottom of the independent cavity and is used for forming an air closed cavity at the bottom of the independent cavity;

5) forming nanopores in the barrier layer, wherein each independent cavity is correspondingly provided with the nanopores;

6) forming a micro-channel structure below the cavity layer, wherein the micro-channel structure is used for injecting a water-phase reaction solution into the independent cavity and injecting an oil-phase liquid seal layer into the lower surface of the cavity layer;

7) and forming an oil phase liquid seal layer on the lower surface of the cavity layer, wherein the oil phase liquid seal layer and the water phase reaction solution in the independent cavity form an oil-water interface so as to seal and isolate the water phase reaction solution in the independent cavity.

12. The method for manufacturing a nanopore detection device based on a heated sealing structure of claim 11, wherein: the method also comprises a step of preparing an electrode structure, wherein the electrode structure comprises a common electrode arranged in the common liquid cavity and independent electrodes arranged in each independent cavity.

13. The method for manufacturing a nanopore detection device based on a heated sealing structure of claim 11, wherein: step 5) the nanopore comprises one of a solid nanopore and a biological nanopore, the barrier layer of the solid nanopore comprises an insulating medium layer, the barrier layer of the biological nanopore comprises one of a lipid molecule layer and a block copolymer molecule layer, the insulating medium layer comprises 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 comprises a phospholipid bilayer.

14. The method for manufacturing a nanopore sensing device based on a heated sealing structure of claim 13, wherein: the method for forming the solid-state nano-pores in the barrier layer comprises the following steps:

forming a conductive metal in the independent cavity;

forming independent electrodes corresponding to each independent cavity on the conductive metal, wherein the independent electrodes expose part of the independent cavities to form a removal window, and a common electrode is manufactured in the common liquid cavity, and the melting temperatures of the independent electrodes and the common electrode are higher than that of the conductive metal;

causing the conductive metal to break down the barrier layer by applying a breakdown voltage to the individual electrodes and a common electrode to simultaneously form nanopores corresponding to each individual cavity in the barrier layer;

and removing the conductive metal from the removal window by heating and melting.

15. The method for manufacturing a nanopore detection device based on a heated sealing structure of claim 14, wherein: the conductive metal comprises one of germanium, tin, indium and bismuth, and the independent electrode and the common electrode are made of one of copper, aluminum, titanium nitride, gold and platinum.

16. The method for manufacturing a nanopore detection device based on a heated sealing structure of claim 13, wherein: the shape of the solid state nanopore comprises one of a cylinder, a cone, a tower, and a funnel.

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