CN115125098A

CN115125098A - Nanopore detection device and method based on molecular sealing layer and heating sealing structure

Info

Publication number: CN115125098A
Application number: CN202110335197.4A
Authority: CN
Inventors: 不公告发明人
Original assignee: Shanghai Jinguan Technology Co ltd
Current assignee: Shanghai Jinguan Technology Co ltd
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2022-09-30

Abstract

The invention provides a nanopore detection device based on a molecular sealing layer and a heating sealing structure and a manufacturing method thereof, wherein the device comprises: the barrier layer is formed with a plurality of nano holes, and a public liquid cavity is arranged above the barrier layer; a cavity layer comprising a plurality of independent cavities; a micro flow channel structure for injecting a solution; the oil phase liquid seal layer and the water phase reaction solution in the independent cavities form an oil-water interface so as to seal and isolate the water phase reaction solution in the respective independent cavities; a lipid molecule sealing layer, wherein a hydrophilic group is dissolved in the aqueous phase reaction solution, and a hydrophobic group is dissolved in the oil phase liquid sealing layer; and the heating sealing structure is used for forming an air sealing cavity at the bottom of the independent cavity. According to the invention, the oil-phase liquid seal layer and the lipid molecule seal layer seal and isolate the water-phase reaction solution in respective independent cavities, so that the double-layer liquid seal effect of the independent cavities is realized, and the air seal cavity is formed at the bottom of the independent cavity through the heating electrode, so that the good liquid seal effect is realized.

Description

Nanopore detection device and method based on molecular sealing layer and heating sealing structure

Technical Field

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

Background

Most of the existing nanopore sequencing technologies use a form of measuring ion current to measure the blocking current generated by the DNA via hole, and the magnitude of the blocking current is different according to the difference of size information and charge information of different bases, so that different bases correspond to different blocking currents, and the sequence information of DNA can be analyzed; the nano-pores are generally arranged on an insulating film, for example, the biological nano-pores are embedded on an insulating lipid bilayer film, and the solid nano-pores are prepared on the solid insulating film through a semiconductor processing technology; the nanopore and insulating film are placed in a dielectric solution (typically KCl solution) that separates the solution into two parts; a driving voltage is applied to both sides of the insulating film, and this driving voltage has 2 functions: on one hand, the voltage can drive charged ions in the salt solution to pass through the nano-pores, and the movement of the charged ions generates ion current of the through-pores; the other side of the driving voltage is used for driving the charged DNA molecules to move to pass through the nano-pores, and the DNA molecules can block the movement of ions in the nano-pores when moving in the nano-pores, so that the intensity of the ionic current can be reduced to form blocking current; because the sizes and the charge information of four bases of DNA are different, the magnitude of the blocking current generated by different bases is different, which is the basic principle of nanopore sequencing.

If the sequencing flux needs to be improved, a large number of nanopores are needed 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, sufficient sealing conditions need to be arranged between the independent electrodes and the solution chambers, salt solutions between the independent chambers cannot leak, otherwise, ion current signals generated in each nanopore can generate leakage current and crosstalk phenomena, adverse phenomena such as noise improvement and cross interference of signals are caused, and accurate interpretation of the signals is influenced.

Disclosure of Invention

In view of the above drawbacks of the prior art, an object of the present invention is to provide a nanopore detection device based on a molecular sealing layer and a heating sealing structure, a manufacturing method thereof, and an application thereof, for solving the problem in the prior art that an ion current signal generated in each nanopore of a nanopore array is prone to leakage current and crosstalk.

To achieve the above and other related objects, the present invention provides a nanopore sensing device based on a molecular sealing layer and a heat sealing structure, the sensing 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 and a lipid molecule 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; the lipid molecule sealing layer is positioned at the oil-water interface and comprises a hydrophilic group and a hydrophobic group, the hydrophilic group is dissolved in the aqueous phase reaction solution, and the hydrophobic group is dissolved in the oil-phase liquid sealing layer; and the heating 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.

Optionally, the nanopore includes one of a solid nanopore and a biological nanopore, a barrier layer of the solid nanopore includes an insulating medium layer, the barrier layer of the biological nanopore includes one of a lipid molecule layer and a block copolymer molecule layer, the insulating medium 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.

Optionally, the solid state nanopore has a shape comprising one of a cylinder, a cone, a tower, and a funnel.

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

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

Optionally, the liquid dispenser further comprises an electrode structure, wherein the electrode structure comprises a common electrode arranged in the common liquid cavity and an independent electrode arranged in each independent cavity.

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 lipid molecule envelope comprises one of a phospholipid molecule, a glycolipid molecule, a diglyceride, a triglyceride, and a glycerophosphate.

Optionally, the hydrophilic group comprises one of hydroxyl, carboxyl, amino, and phosphoric acid, and the hydrophobic group comprises an alkane chain.

Optionally, the heating electrode comprises a ring-shaped heating electrode surrounding the bottom of the independent cavity.

Optionally, the detection apparatus is used for detecting a DNA sequence, and a driving voltage is applied to two sides of the nanopore to drive the ions in the aqueous reaction solution to move to generate a current, and simultaneously a DNA strand is driven to pass through the nanopore, the DNA strand blocks the ion movement 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.

The invention also provides an application method of the nanopore detection device based on the molecular sealing layer and the heating sealing structure, which comprises the following steps: 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 dissolved with lipid molecules to the lower surface of the cavity layer based on the micro-channel structure, wherein the oil phase liquid seal layer and an aqueous phase reaction solution in the independent cavity form an oil-water interface, the lipid molecules self-assemble at the oil-water interface to form a lipid molecule seal layer, the lipid molecule seal layer comprises a hydrophilic group and a hydrophobic group, the hydrophilic group is dissolved in the aqueous phase reaction solution, and the hydrophobic group is dissolved in the oil phase liquid seal layer, so as to seal and isolate the aqueous phase reaction solution in the respective 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.

The invention also provides a manufacturing method of the nanopore detection device based on the molecular sealing layer and the heating sealing structure, and the manufacturing method comprises 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) forming an oil phase liquid seal layer dissolved with lipid molecules 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, the lipid molecules are self-assembled at the oil-water interface to form a lipid molecule seal layer, the lipid molecule seal layer comprises a hydrophilic group and a hydrophobic group, the hydrophilic group is dissolved in the water phase reaction solution, and the hydrophobic group is dissolved in the oil phase liquid seal layer so as to seal and isolate the water phase reaction solution in respective independent cavity.

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

Optionally, the nanopore of step 5) comprises one of a solid-state nanopore and a biological nanopore, the barrier layer of the solid-state 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.

Optionally, the method for forming the solid-state nanopore in the barrier layer includes the steps of: 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.

Optionally, the conductive metal comprises one of cadmium, tin, indium and bismuth, and the material of the individual electrode and the common electrode comprises one of copper, aluminum, titanium nitride, gold and platinum.

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

the invention provides a nanopore detection device based on a molecular seal layer and a heating seal structure, wherein after a public liquid cavity and an independent cavity are filled with a water phase solution, an oil phase liquid seal layer is injected through a micro flow channel, the oil phase liquid seal layer can squeeze the water phase solution in the micro flow channel away and replace the water phase solution, the lower surface of a cavity body layer is paved, under the action of surface tension, the water phase solution in the independent cavity cannot be replaced by the oil phase liquid seal layer but is sealed in the independent cavity by the oil phase liquid seal layer to form an oil-water interface, the water phase reaction solution is sealed and isolated in the respective independent cavity, salt solution cross leakage which may occur between the independent cavities is prevented, and the effect of sealing the independent cavities is realized.

The invention forms the lipid molecular sealing layer by self-assembly at the oil-water interface, the existence of the lipid molecular sealing layer can enhance the liquid sealing effect, and the lipid molecular sealing layer and the oil phase liquid sealing layer jointly realize double-layer liquid sealing of an independent cavity, thereby further avoiding liquid leakage in the reaction process and realizing better effect of sealing the liquid cavity for the independent cavity.

According to the invention, the heating electrode is arranged at the bottom of the independent cavity, and the bottom of the independent cavity is heated after being electrified, so that an air sealing cavity is formed at the bottom of the independent cavity, and the excellent liquid sealing effect is realized together with the oil phase liquid sealing layer and the lipid molecule sealing layer.

According to the invention, the conductive metal is formed in the independent cavities, the nano holes 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, so that the preparation of the nano hole array with high alignment precision can be realized, the preparation cost of the nano hole array can be effectively reduced, and the preparation method has the advantages of simple and stable process.

Drawings

Fig. 1 is a schematic structural diagram of a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to an embodiment of the present invention.

Fig. 2 to 5 are schematic diagrams illustrating a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to an embodiment of the invention.

Fig. 6 and 7 are schematic structural views illustrating a lipid molecule sealing layer of a nanopore sensing device based on a molecule sealing layer and a heating sealing structure according to an embodiment of the invention.

Fig. 8 is a schematic flow chart illustrating steps of a method for applying a nanopore sensing device based on a molecular sealing layer and a heating sealing structure according to an embodiment of the present invention.

Fig. 9 is a schematic flow chart illustrating steps of a method for manufacturing a nanopore sensing device based on a molecular sealing layer and a heating sealing structure according to an embodiment of the invention.

Description of the element reference

101 cavity layer

102 independent cavity

103 barrier layer

104 nanopore

105 micro-channel structure

106 oil phase liquid seal layer

107 oil-water interface

108 common liquid chamber

109 independent electrode

110 common electrode

111 lipid molecule envelope

1111 hydrophilic group

1112 hydrophobic groups

112 heating electrode

113 air-tight chamber

S11-S13

S21-S26

Detailed Description

The following embodiments of the present invention are provided by way of specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

As in the detailed description of the embodiments of the present invention, the cross-sectional views illustrating the device structures are not partially enlarged in general scale for convenience of illustration, and the schematic views are only examples, which should not limit the scope of the present invention. In addition, the three-dimensional dimensions of length, width and depth should be included in the actual fabrication.

Spatially relative terms, such as "under," "below," "lower," "below," "over," "upper," and the like, may be used herein for convenience in describing the relationship of one element or feature to another element or feature illustrated in the figures. It will be understood that these terms of spatial relationship are intended to encompass other orientations of the device in use or operation in addition to the orientation 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, a structure described as having a first feature "on" a second feature 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 in between the first and second features, such that the first and second features may not be in direct contact.

It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the present invention, and the drawings only show the components related to the present invention rather than being drawn according to the number, shape and size of the components in actual implementation, and the type, quantity and proportion of each component in actual implementation may be changed arbitrarily, and the layout of the components may be more complicated.

As shown in fig. 1 to 7, the present embodiment provides a nanopore detection device based on a molecular sealing layer and a heating sealing structure, the detection device comprising: the liquid-liquid separation device comprises a common liquid cavity 108, a barrier layer 103, a cavity layer 101, a micro-channel structure 105, an oil-phase liquid seal layer 106 and a lipid molecule seal layer 111.

As shown in fig. 1, a plurality of nano-pores 104 are formed in the barrier layer 103 to penetrate the barrier layer 103.

The nanopore 104 comprises one of a solid nanopore 104 and a biological nanopore 104, the barrier layer 103 of the solid nanopore 104 comprises an insulating medium layer, the barrier layer 103 of the biological nanopore 104 comprises one of a lipid molecular layer and a block copolymer molecular 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 molecular layer comprises 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.

As shown in FIG. 2, in one embodiment, the solid-state nanopore 104 has a cylindrical shape, and the diameter of the nanopore 104 may be 0.1 to 99nm, and preferably, the diameter of the nanopore 104 is 1 to 5 nm.

In another embodiment, as shown in FIG. 3, the solid-state nanopore 104 is tapered, and the tapered nanopore 104 has a minimum pore size, which may be 0.1 to 99nm, preferably 1 to 5 nm. Will solid-state nanopore 104 sets up to the toper, can effectively reduce on the one hand the actual thickness of solid-state nanopore 104 (the thickness of the corresponding barrier layer 103 in minimum aperture department is less promptly), can avoid nanopore 104 to be blockked up completely when guaranteeing measurement accuracy, guarantee the flow of DNA in the aqueous phase reaction solution, on the other hand, can reduce the actual migration distance of DNA in nanopore 104, improve and detect the precision.

In yet another embodiment, as shown in fig. 4, the solid-state nanopore 104 has a tower shape, the tower-shaped nanopore 104 comprises two or more circular holes with different diameters connected in sequence, and the tower-shaped nanopore 104 has a minimum pore diameter, which may be 0.1-99 nm, preferably 1-5 nm. Will solid-state nanopore 104 sets up to the turriform, can effectively reduce on the one hand the actual thickness of solid-state nanopore 104 (the thickness of the barrier layer 103 that corresponds of minimum aperture department is less promptly), can avoid nanopore 104 to be blockked up completely when guaranteeing measurement accuracy, guarantees the flow of DNA in the aqueous phase reaction solution, and on the other hand can reduce the actual migration distance of DNA in nanopore 104, improves and detects the precision.

In yet another embodiment, as shown in FIG. 5, the solid-state nanopore 104 has a funnel shape, the funnel-shaped nanopore 104 includes two opposite tapered pores connected together, and the funnel-shaped nanopore 104 has a minimum pore size, which may be 0.1-99 nm, preferably 1-5 nm. Will solid-state nanopore 104 sets up to the infundibulate, can effectively reduce on the one hand the actual thickness of solid-state nanopore 104 (the thickness of the corresponding barrier layer 103 in minimum aperture department is less promptly), can avoid nanopore 104 to be blockked up completely when guaranteeing measurement accuracy, guarantee the flow of DNA in the aqueous phase reaction solution, on the other hand, can reduce the actual migration distance of DNA in nanopore 104, improve and detect the precision.

As shown in fig. 1, the common liquid chamber 108 is located above the barrier layer 103 and is used for carrying an aqueous phase reaction solution, and the aqueous phase reaction solution in the common liquid chamber 108 can be directly injected or injected through a micro flow channel structure.

As shown in fig. 1, the cavity layer 101 is located below 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 cavity layer 101 can be made of silicon dioxide and the like, the independent cavities 102 are etched in the silicon dioxide in a photoetching-etching mode, the independent cavities 102 can be cylindrical cavities, the diameter of each cylindrical cavity is 1-1000 micrometers, and the interval between every two adjacent cylindrical cavities is 2-5000 micrometers. Of course, in other embodiments, the shape of the independent cavity may be other shapes such as an ellipse, a polygon, etc., and is not limited to the examples listed herein.

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 arranged in a periodic array, so as to improve the flux and efficiency of detection.

As shown in fig. 1, the micro flow channel structure 105 is located below the cavity layer 101, and is used for injecting an aqueous phase reaction solution into the independent cavity 102, and injecting an oil phase liquid seal layer 106 and a lipid molecule seal layer 111 into the lower surface of the cavity layer 101.

As shown in fig. 1, the oil phase liquid seal layer 106 is located on the lower surface of the cavity layer 101, and the oil phase liquid seal layer 106 and the aqueous phase reaction solution in the independent cavity 102 form an oil-water interface 107 to seal and isolate the aqueous phase reaction solution in the respective independent cavity 102. After the common liquid cavity 108 and the independent cavities 102 are filled with the aqueous phase solution, the oil phase liquid seal layer 106 is injected through the micro-channel, and the oil phase liquid seal layer 106 is paved on the lower surface of the cavity layer 101, as shown in fig. 1, under the action of surface tension, the aqueous phase solution in the independent cavities 102 is not replaced by the oil phase liquid seal layer 106, but is sealed in the independent cavities 102 by the oil phase liquid seal layer 106, so as to form an oil-water interface 107, so that the aqueous phase reaction solution is sealed and isolated in the respective independent cavities 102, thereby preventing the salt solution from cross leakage possibly occurring between the independent cavities 102, and realizing the effect of sealing the independent cavities 102.

As shown in fig. 6 and 7, the lipid molecule sealing layer 111 is located at the oil-water interface 107, the lipid molecule sealing layer 111 includes a hydrophilic group 1111 and a hydrophobic group 1112, the hydrophilic group 1111 is dissolved in the aqueous phase reaction solution, and the hydrophobic group 1112 is dissolved in the oil phase liquid sealing layer. The lipid molecule sealing layer 111 comprises amphiphilic molecules, when the amphiphilic molecules are independently dissolved in an aqueous solution, the amphiphilic molecules can slowly perform self-assembly on the surface of the aqueous solution, hydrophilic groups 1111 are dissolved in water, and hydrophobic groups 1112 are distributed in the air; self-assembly also occurs when the amphiphilic molecule is dissolved alone in the oil phase organic solvent, with its hydrophobic groups 1112 (such as alkane chains) dissolved in the oil phase and its hydrophilic groups 1111 distributed in the air; in the mixed liquid of the oil phase and the water phase, the amphiphilic molecule can perform self-assembly action at an oil-water interface, the hydrophilic group 1111 is dissolved in the water solution, and the hydrophobic group 1112 is dissolved in the oil phase solvent. In this embodiment, the lipid molecule envelope includes one of phospholipid molecules, glycolipid molecules, diglycerides, triglycerides, and glycerophosphates. As an example, as shown in fig. 7, the hydrophilic group 1111 includes one of hydroxyl, carboxyl, amino, and phosphoric acid, and the hydrophobic group 1112 includes an alkane chain, and in this embodiment, the number of the alkane chain is 1 to 3.

The invention forms the lipid molecule sealing layer in the oil-water interface by self-assembly, the existence of the lipid molecule sealing layer can enhance the liquid sealing effect, and the lipid molecule sealing layer and the oil phase liquid sealing layer jointly realize the double-layer liquid sealing of the independent cavity, further avoid the liquid leakage in the reaction process and realize the better effect of sealing the liquid cavity for the independent cavity.

As shown in fig. 1, the heating and sealing structure includes a heating electrode 112 located at the bottom of the independent cavity 102 for forming an air-tight cavity 113 at the bottom of the independent cavity 102.

The heating electrode 112 includes an annular heating electrode surrounding the bottom of the independent cavity 102, and the heating electrode 112 of this embodiment employs an annular heating electrode surrounding the bottom of the independent cavity 102, which is beneficial to forming an air-tight cavity 113, so that the air-tight cavity 113 completely covering the entire bottom of the independent cavity 102 is easily formed, and the sealing and isolation of the independent cavity 102 can be very effectively realized. Of course, in other embodiments, the heating electrode 112 may also be a block electrode, and two or more block electrodes are uniformly distributed at the bottom of the independent cavity 102 to form an air-tight cavity 113 at the bottom of the independent cavity 102, and it should be noted that the configuration of the heating electrode 112 is not limited to the above-mentioned examples, and may be selected according to actual requirements. As shown in fig. 1, the detection apparatus further includes an electrode structure including a common electrode 110 disposed in the common liquid chamber 108 and an individual electrode 109 disposed in each of the individual chamber bodies 102.

The detection device is used for detecting a DNA sequence, a driving voltage is applied to two sides of the nanopore 104 to drive ions in the aqueous phase reaction solution to move to generate current, a DNA chain is driven to pass through the nanopore 104 at the same time, the DNA chain blocks the ions when passing through the nanopore 104 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.

As shown in fig. 8, the present invention further provides an application method of a nanopore detection device based on a molecular sealing layer and a heating sealing structure, comprising:

step 1) S11, injecting an aqueous phase reaction solution into the independent cavity 102 based on the micro flow channel structure 105, the aqueous phase reaction solution containing the DNA to be detected and a dielectric solution, the dielectric solution being, for example, a potassium chloride (KCl) solution;

step 2) S12, injecting an oil-phase liquid seal layer 106 dissolved with lipid molecules to the lower surface of the cavity layer 101 based on the micro flow channel structure 105, where the oil-phase liquid seal layer 106 and the aqueous phase reaction solution in the independent cavity 102 form an oil-water interface 107, the lipid molecules self-assemble at the oil-water interface to form a lipid molecule seal layer 111, the lipid molecule seal layer 111 includes a hydrophilic group 1111 and a hydrophobic group 1112, the hydrophilic group 1111 is dissolved in the aqueous phase reaction solution, and the hydrophobic group 1112 is dissolved in the oil-phase liquid seal layer 106, so as to seal and isolate the aqueous phase reaction solution in the respective independent cavity 102, thereby achieving a "double-layer liquid seal" effect;

step 3) S13, heating by the heating electrode 112 to form an air-tight cavity 113 at the bottom of the independent cavity 102, where the air-tight cavity 113 completely covers the bottom of the independent cavity 102;

and 4) S14, applying a driving voltage on two sides of the nanopore 104 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 104, wherein the DNA chain blocks the ions when passing through the nanopore 104 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.

As shown in fig. 1 to 7 and 9, the present invention further provides a method for manufacturing a nanopore detection device based on a molecular sealing layer and a heating sealing structure, wherein the method comprises the steps of:

as shown in fig. 1 and 9, step 1) S21 is performed first, 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 barrier layer 103 includes an insulating dielectric layer or a lipid molecular layer, the insulating dielectric layer includes one of silicon nitride, silicon dioxide, aluminum oxide, hafnium oxide, zinc oxide, titanium oxide, boron nitride, molybdenum disulfide, and graphene, the lipid molecular layer includes a phospholipid bilayer, specifically, according to a difference of subsequently formed nanopores 104, 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 molecular layer and a block copolymer molecular layer.

As shown in fig. 1 and 9, step 2) S22 is then performed to etch the substrate to form the common liquid chamber 108.

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

The material of the cavity layer 101 can be silicon dioxide and the like, the independent cavities 102 are etched in the silicon dioxide in a photoetching-etching mode, the independent cavities 102 can be cylindrical cavities, the diameter of each cylindrical cavity is 1-1000 micrometers, and the interval between every two adjacent cylindrical cavities is 2-5000 micrometers. Of course, in other embodiments, the shape of the independent cavity may be other shapes such as an ellipse, a polygon, etc., and is not limited to the examples listed herein.

As shown in fig. 1 and 9, step 4) S24 is then performed to form a heating and sealing structure at the bottom of the independent cavity 102, where the heating and sealing structure includes a heating electrode 112 located at the bottom of the independent cavity 102 for forming an air-tight cavity 113 at the bottom of the independent cavity 102.

In this embodiment, the heating electrode 112 includes an annular heating electrode surrounding the bottom of the independent cavity 102, and the heating electrode 112 of this embodiment adopts the annular heating electrode surrounding the bottom of the independent cavity 102, which can facilitate the formation of the air-tight cavity 113, easily form the air-tight cavity 113 completely covering the entire bottom of the independent cavity 102, and can very effectively realize the sealing and isolation of the independent cavity 102. Of course, in other embodiments, the heating electrode 112 may also be a block electrode, and two or more block electrodes are uniformly distributed at the bottom of the independent cavity 102 to form an air-tight cavity 113 at the bottom of the independent cavity 102, and it should be noted that the configuration of the heating electrode 112 is not limited to the above-mentioned examples, and may be selected according to actual requirements.

As shown in fig. 1 and 9, step 5) S25 is then performed to form nanopores 104 in the barrier layer 103, wherein each of the independent cavities 102 is configured with the nanopores 104.

The nanopore 104 includes one of a solid-state nanopore 104 and a biological nanopore 104, in this embodiment, the nanopore 104 is the solid-state nanopore 104, and the solid-state nanopore 104 has one of a cylindrical shape, a conical shape, a tower shape, and a funnel shape.

In another embodiment, as shown in fig. 4, the solid-state nanopore 104 has a tower shape, the tower-shaped nanopore 104 includes two or more circular holes with different diameters connected in sequence, and the tower-shaped nanopore 104 has a minimum pore size, which may be 0.1 to 99nm, preferably 1 to 5 nm. Will solid-state nanopore 104 sets up to the turriform, can effectively reduce on the one hand the actual thickness of solid-state nanopore 104 (the thickness of the barrier layer 103 that corresponds of minimum aperture department is less promptly), can avoid nanopore 104 to be blockked up completely when guaranteeing measurement accuracy, guarantees the flow of DNA in the aqueous phase reaction solution, and on the other hand can reduce the actual migration distance of DNA in nanopore 104, improves and detects the precision.

In yet another embodiment, as shown in fig. 5, the solid-state nanopore 104 has a funnel shape, the funnel-shaped nanopore 104 includes two opposing tapered pores connected together, and the funnel-shaped nanopore 104 has a minimum pore size, which may be 0.1 to 99nm, preferably 1 to 5 nm. Will solid-state nanopore 104 sets up to the infundibulate, can effectively reduce on the one hand the actual thickness of solid-state nanopore 104 (the thickness of the corresponding barrier layer 103 in minimum aperture department is less promptly), can avoid nanopore 104 to be blockked up completely when guaranteeing measurement accuracy, guarantee the flow of DNA in the aqueous phase reaction solution, on the other hand, can reduce the actual migration distance of DNA in nanopore 104, improve and detect the precision.

In this embodiment, the method for forming the solid-state nano-pores 104 in the barrier layer 103 includes the steps of:

step 5-1) forming conductive metal in the independent cavity 102; forming an individual electrode 109 corresponding to each individual cavity 102 on the conductive metal, wherein the individual electrode 109 exposes a portion of the individual cavity 102 to form a removal window, and a common electrode 110 is formed in the common liquid chamber 108, wherein the melting temperature of the individual electrode 109 and the common electrode 110 is higher than the melting temperature of the conductive metal;

step 5-2) applying breakdown voltage to the independent electrode 109 and the common electrode to enable the conductive metal to break down the barrier layer 103 so as to simultaneously form the nano holes 104 corresponding to each independent cavity 102 in the barrier layer 103;

and 5-3) removing the conductive metal from the removal window in a heating melting mode.

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

According to the invention, conductive metal is formed in the independent cavities 102, the nano holes 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, so that the preparation of the nano hole 104 array with high alignment precision can be realized, the preparation cost of the nano hole 104 array can be effectively reduced, and the preparation method has the advantages of simple and stable process.

As shown in fig. 1 and 9, step 6) S26 is performed to form a micro flow channel structure 105 below the cavity layer 101, where the micro flow channel structure 105 is used to inject the aqueous phase reaction solution into the independent cavity 102 and the common liquid cavity 108, and to inject the oil phase liquid seal layer 106 into the lower surface of the cavity layer 101.

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

As shown in fig. 1, under the action of surface tension, the aqueous phase solution in the independent cavities 102 is not replaced by the oil phase liquid seal layer 106, but is enclosed in the independent cavities 102 by the oil phase liquid seal layer 106 to form an oil-water interface 107, so as to enclose and isolate the aqueous phase reaction solution in the respective independent cavities 102, thereby preventing the salt solution from leaking between the independent cavities 102, and achieving the effect of enclosing the independent cavities 102.

The concentration range of the lipid molecules dissolved in the oil phase liquid seal layer 106 is 1 μ M to 10M, and in this embodiment, the concentration range of the lipid molecules dissolved in the oil phase liquid seal layer 106 is 100nM to 1000 mM.

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

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A nanopore detection device based on a molecular seal and a heating seal structure is characterized in that the detection device comprises:

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 and a lipid molecule 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;

the lipid molecule sealing layer is positioned at the oil-water interface and comprises a hydrophilic group and a hydrophobic group, the hydrophilic group is dissolved in the aqueous phase reaction solution, and the hydrophobic group is dissolved in the oil-phase liquid sealing layer;

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 detection device based on the molecular sealing layer and the heating 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 detection device based on the molecular sealing layer and the heating 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 detection device based on the molecular sealing layer and the heating sealing structure of claim 1, wherein: the minimum aperture of the nanopore is 0.1-99 nm.

5. The nanopore detection device based on the molecular sealing layer and the heating 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 the molecular sealing layer and the heating 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 the molecular sealing layer and the heating 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 lipid molecule sealing layer comprises one of phospholipid molecules, glycolipid molecules, diglyceride, triglyceride and glycerophosphate.

9. The nanopore detection device based on the molecular sealing layer and the heating sealing structure of claim 1, wherein: the hydrophilic group comprises one of hydroxyl, carboxyl, amino and phosphoric acid, and the hydrophobic group comprises an alkane chain.

10. The nanopore sensing 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.

11. The nanopore detection device based on the molecular sealing layer and the heating 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.

12. The application method of the nanopore detection device based on the molecular sealing layer and the heating sealing structure as claimed in any one of claims 1 to 11, 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 dissolved with lipid molecules to the lower surface of the cavity layer based on the micro-channel structure, wherein the oil phase liquid seal layer and an aqueous phase reaction solution in the independent cavity form an oil-water interface, the lipid molecules self-assemble at the oil-water interface to form a lipid molecule seal layer, the lipid molecule seal layer comprises a hydrophilic group and a hydrophobic group, the hydrophilic group is dissolved in the aqueous phase reaction solution, and the hydrophobic group is dissolved in the oil phase liquid seal layer, so as to seal and isolate the aqueous phase reaction solution in the respective 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.

13. A manufacturing method of a nanopore detection device based on a molecular sealing layer and 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 sealing structure at the bottom of the independent cavity, wherein 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;

5) forming a nanopore in the barrier layer, wherein each independent cavity is correspondingly configured with the nanopore;

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) forming an oil phase liquid seal layer dissolved with lipid molecules 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, the lipid molecules are self-assembled at the oil-water interface to form a lipid molecule seal layer, the lipid molecule seal layer comprises a hydrophilic group and a hydrophobic group, the hydrophilic group is dissolved in the water phase reaction solution, and the hydrophobic group is dissolved in the oil phase liquid seal layer so as to seal and isolate the water phase reaction solution in respective independent cavity.

14. The method for manufacturing a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 13, 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.

15. The method for manufacturing a nanopore sensing device based on a molecular sealing layer and a heating sealing structure according to claim 13, wherein: step 5) the nanopore comprises one of a solid nanopore and a biological nanopore, the 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.

16. The method for manufacturing a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 15, 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 breakdown voltages to the individual electrodes and a common electrode to simultaneously form nanopores in the barrier layer corresponding to each individual cavity;

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

17. The method for manufacturing a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 16, wherein: the conductive metal comprises one of cadmium, tin, indium and bismuth, and the independent electrode and the common electrode are made of one of copper, aluminum, titanium nitride, gold and platinum.

18. The method for manufacturing a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 15, wherein: the shape of the solid state nanopore comprises one of a cylinder, a cone, a tower, and a funnel.

19. The method for manufacturing a nanopore sensing device based on a molecular sealing layer and a heating sealing structure according to claim 13, wherein: the lipid molecule sealing layer comprises one of phospholipid molecules, glycolipid molecules, diglyceride, triglyceride and glycerophosphate.

20. The method for manufacturing a nanopore detection device based on a molecular sealing layer and a heating sealing structure according to claim 13, wherein: the hydrophilic group comprises one of hydroxyl, carboxyl, amino and phosphoric acid, and the hydrophobic group comprises an alkane chain.