WO2022227080A1 - 孔阵列层结构、芯片装置、纳米孔测序装置和成膜方法以及用途 - Google Patents

孔阵列层结构、芯片装置、纳米孔测序装置和成膜方法以及用途 Download PDF

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WO2022227080A1
WO2022227080A1 PCT/CN2021/091732 CN2021091732W WO2022227080A1 WO 2022227080 A1 WO2022227080 A1 WO 2022227080A1 CN 2021091732 W CN2021091732 W CN 2021091732W WO 2022227080 A1 WO2022227080 A1 WO 2022227080A1
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Prior art keywords
accommodating cavity
layer structure
layer
hole array
polar medium
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PCT/CN2021/091732
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English (en)
French (fr)
Inventor
张喆
任世龙
宋璐
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成都齐碳科技有限公司
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Priority to EP21938554.9A priority Critical patent/EP4310487A4/en
Priority to PCT/CN2021/091732 priority patent/WO2022227080A1/zh
Publication of WO2022227080A1 publication Critical patent/WO2022227080A1/zh
Priority to US18/384,519 priority patent/US20240053297A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present application relates to the technical field of biological detection, and in particular, to a pore array layer structure, a chip device, a nanopore sequencing device, and a film forming method and application.
  • Nanopore detection technology has a wide range of applications in the fields of protein detection, gene sequencing and nanoparticle characterization.
  • nanopores are arranged in the membrane of the gene sequencing device.
  • deoxyribonucleic acid (DeoxyriboNucleic Acid, DNA) molecules pass through the nanopore, due to the difference in the shape and size of different bases, it is different from the inside of the nanopore.
  • the detection molecule reacts specifically to cause a change in resistance.
  • There is a constant voltage on both sides of the nanopore which can detect the change of the current of different bases passing through the nanopore, thus reflecting the base arrangement of the DNA molecule passing through the nanopore.
  • the stability of the thin film of the gene sequencing device is crucial to the accuracy of detection, but at present the stability of the thin film is not good, resulting in poor detection accuracy.
  • the present application provides a pore array layer structure, a chip device, a nanopore sequencing device, a film-forming method and uses, aiming at solving the problem of poor film stability and improving the film-forming stability.
  • an embodiment of the present application provides a hole array layer structure, which is used to form a film-forming space with a substrate, and the film-forming space is used to form a film layer.
  • the hole array layer structure includes: a plurality of hole units arranged in an array, the hole units pass through the hole array layer structure, the hole unit has a first accommodating cavity and a second accommodating cavity that are in axial communication, and the first accommodating cavity is configured to be connected to the substrate connected, at least one first accommodating cavity is communicated with other first accommodating cavities.
  • At least one second accommodating cavity communicates with other second accommodating cavities.
  • the hole array layer structure includes a first confinement layer and a second confinement layer, the first accommodating cavity is disposed in the first confinement layer and penetrates through the first confinement layer, and the second accommodating cavity It is arranged in the second limiting layer and penetrates through the second limiting layer.
  • the second limiting layer includes a plurality of second subunits arranged in an array, and the second accommodating cavity is disposed in the second subunit and penetrates through the second subunit.
  • the second limiting layer includes a plurality of third subunits arranged in an array, the third subunits include a third subaccommodating cavity, and the second subunits are located in the third subaccommodating cavity Inside, there is a gap between the second subunit and the third subunit.
  • a plurality of first grooves are defined on the outer side of the second subunit.
  • the first confinement layer includes at least one first channel, and the first channel communicates every adjacent at least two first accommodating cavities.
  • the second limiting layer includes at least one second channel, and the second channel communicates every adjacent at least two second accommodating cavities.
  • the first confinement layer includes at least one first communication groove, the first communication groove extending radially outward of the first accommodating cavity, the first communication groove facing away from the first accommodating cavity one end is closed.
  • the first confinement layer includes at least one first communication groove, and the first communication groove penetrates the first confinement layer.
  • the second limiting layer includes at least one second communication groove extending radially outward of the second accommodating cavity, the second communicating groove facing away from the second accommodating cavity one end is closed.
  • the second confinement layer includes at least one second communication groove, and the second communication groove penetrates the second confinement layer.
  • the first limiting layer includes at least one first communication groove, the at least one first communication groove forms a third accommodating cavity, and the diameter or width of the third accommodating cavity is larger than that of the second accommodating cavity diameter or width.
  • the diameter or width of the first accommodating cavity is smaller than or equal to the diameter or width of the second accommodating cavity.
  • an embodiment of the present application provides a chip device, which includes, for example, a substrate; the hole array layer structure according to the embodiment of the first aspect, the hole array layer structure is located on the substrate, and the hole array layer structure includes an array arrangement a plurality of hole units, the second accommodating cavity of the hole unit is located on the side of the first accommodating cavity of the hole unit away from the substrate; and a membrane layer, the membrane layer is located in the plurality of hole units, and the membrane layer is configured to detect the test to be tested sample.
  • an embodiment of the present application provides a method for forming a film, including: arranging a first non-polar medium in a hole array layer structure, forming a precoat film layer on the surface of the hole array layer structure; flowing through the hole array layer structure, replacing at least part of the first non-polar medium; flowing a second non-polar medium through the hole array layer structure, replacing at least part of the first polar medium, wherein the second non-polar medium
  • the polar medium comprises an amphiphilic molecular material; flowing a second polar medium through the pore array layer structure, replacing at least part of the first non-polar medium, and in a space between the first polar medium and the second polar medium;
  • the interface forms a membrane layer, wherein the membrane layer contains an amphiphilic molecular material.
  • the embodiments of the present application provide a nanopore sequencing device, including the pore array layer structure of the first aspect of the present application, the chip device of the second aspect of the present application, or the third aspect of the present application.
  • the film layer prepared by the film forming method.
  • the fifth aspect is the use of the hole array layer structure of the embodiment of the first aspect of the present application, the chip device of the embodiment of the second aspect of the present application, or the film layer prepared by the film-forming method of the embodiment of the third aspect of the present application in characterizing an analyte
  • the analyte includes: a biopolymer, and the biopolymer is selected from one of polynucleotides, polypeptides, polysaccharides and lipids.
  • the biopolymer is a polynucleotide comprising DNA and/or RNA.
  • the hole array layer structure includes a plurality of hole units arranged in an array, and a membrane layer is formed in the hole units; each hole unit includes a first accommodating cavity and a second accommodating cavity that communicate with each other , at least one first accommodating cavity is communicated with other first accommodating cavities, and the first non-polar medium located in the first accommodating cavity can diffuse and flow in the adjacent first accommodating cavity, so as to realize the first non-polar medium in different first accommodating cavities.
  • the series complementation between the accommodating cavities and the uniform distribution on the one hand, it can ensure the uniformity of pre-coating and provide a better film forming basis for film formation; on the other hand, it can ensure the stability of the film layer during the film forming stage and tandem complement.
  • FIG. 1 is a perspective view of a hole array layer structure provided according to an embodiment of the present application.
  • Fig. 2 is a schematic diagram of film formation
  • FIG. 3 is an exploded perspective view of a hole position limiting layer provided according to an embodiment of the present application.
  • FIG. 4 is a top view of a hole position defining layer provided according to an embodiment of the present application.
  • FIG. 5 is an exploded perspective view of a hole-limiting layer provided according to a first alternative embodiment of the present application.
  • FIG. 6 is a top view of a hole-limiting layer provided according to a first alternative embodiment of the present application.
  • FIG. 7 is a top view of a second defining layer provided according to a first alternative embodiment of the present application.
  • FIG. 8 is a perspective view of a hole position defining layer provided according to a second alternative embodiment of the present application.
  • FIG. 9 is an exploded perspective view of a hole position defining layer provided according to a second alternative embodiment of the present application.
  • FIG. 10 is a top view of a hole-limiting layer provided according to a second alternative embodiment of the present application.
  • FIG. 11 is an exploded perspective view of a hole position defining layer provided according to a third alternative embodiment of the present application.
  • FIG. 12 is a top view of a hole defining layer provided according to a third alternative embodiment of the present application.
  • FIG. 13 is one of the top views of the second defining layer provided according to the third alternative embodiment of the present application.
  • FIG. 14 is the second top view of the second defining layer provided according to the third alternative embodiment of the present application.
  • FIG. 15 is the third top view of the second defining layer provided according to the third alternative embodiment of the present application.
  • FIG. 16 is an exploded perspective view of a hole-limiting layer provided according to a fourth alternative embodiment of the present application.
  • FIG. 17 is a top view of a hole defining layer provided according to a fourth alternative embodiment of the present application.
  • FIG. 18 is a top view of a second defining layer provided according to a fourth alternative embodiment of the present application.
  • Fig. 19 is the A-A sectional schematic diagram of Fig. 18;
  • FIG. 20 is a top view of a second defining layer provided according to a fifth alternative embodiment of the present application.
  • 21 is a schematic structural diagram of a chip device provided according to an embodiment of the present application.
  • Figure 22 shows a schematic diagram of an electrical characterization test
  • FIG. 23 is an electrical characterization diagram of a chip device background before film formation provided according to an embodiment of the present application.
  • FIG. 25 is an electrical characterization diagram of a chip device provided according to an embodiment of the present application.
  • 26 is a schematic diagram of a film forming process provided according to an embodiment of the present application.
  • Figure 27 is a schematic diagram of the film layer structure.
  • FIG. 1 is a schematic structural diagram of a hole array layer structure provided according to an embodiment of the present application.
  • FIG. 2 is a schematic diagram of film formation provided according to an embodiment of the present application.
  • the hole array layer structure 10 provided in the embodiment of the present application can be used to form a film formation space with the substrate 20 , and the film formation space is used to form the film layer 30 .
  • the hole array layer structure 10 includes a plurality of hole units 13 arranged in an array, and each hole unit 13 penetrates the hole array layer structure 10 .
  • Each hole unit 13 includes a first accommodating cavity 131 and a second accommodating cavity 132 in axial communication.
  • the first accommodating cavity 131 is configured to be connected to the substrate 20 .
  • the first accommodating cavity 131 penetrates the side of the hole array layer structure 10 close to the substrate 20, the second accommodating cavity 132 penetrates the side of the hole array layer structure 10 away from the substrate 20, at least one first accommodating cavity 131 and other first accommodating chambers
  • the cavity 131 communicates.
  • the membrane layer 30 is formed in the hole unit 13 .
  • the film forming step may include step S200 , step 300 , step 400 and step 500 .
  • step S200 the first non-polar medium 31 is arranged in the hole array layer structure 10 , and a precoat film layer is formed on the surface of the hole array layer structure 10 .
  • step S300 the first polar medium 32 is flowed through the hole array layer structure 10 to replace at least part of the first non-polar medium 31 .
  • step S400 a second non-polar medium 33 is flowed through the hole array layer structure 10 to replace at least part of the first polar medium 32, wherein the second non-polar medium 33 includes an amphiphilic molecular material.
  • step S500 the second polar medium 34 is flowed through the hole array layer structure 10 , replacing at least part of the second non-polar medium 33 , and between the first polar medium 32 and the second polar medium 34 .
  • the interface forms a membrane layer 30, and the membrane layer 30 includes an amphiphilic molecular material.
  • the hole array layer structure 10 includes a plurality of hole units 13 arranged in an array, and a membrane layer 30 is formed in the hole units 13;
  • the cavity 131 and the second accommodating cavity 132, at least one first accommodating cavity 131 communicates with other first accommodating cavities 131, and the first non-polar medium 31 located in the first accommodating cavity 131 can be in the adjacent first accommodating cavity 131 Diffusion flow, and a pre-coating film layer is formed on the surface of the hole unit 13 to realize the series complementation and uniform distribution of the first non-polar medium 31 between different first accommodating cavities 131; on the one hand, it can ensure the uniformity of pre-coating It provides a better film-forming basis for film formation.
  • the solvent of the film layer 30 is the second non-polar medium 33, and the second non-polar medium 33 and the first non-polar medium 31 have good compatibility, so the phase between the film layer 30 and the pre-coating film layer is good. Capacitance is good; in the film forming stage, the film layer 30 can flow complementarily with the film layer 30 of the adjacent first accommodating cavity 131 through the pre-coating film layer, which can ensure the stability and series complementation of the film layer 30 .
  • At least one second accommodating cavity 132 communicates with other second accommodating cavities 132 .
  • the first non-polar medium 31 can diffuse and flow between the adjacent second accommodating cavities 132 , so as to realize the series complementation of the first non-polar medium 31 between different second accommodating cavities 132 , and further ensure the stability of the membrane layer 30 . Sex and tandem complementarity.
  • the film layer 30 When the film layer 30 is located at the first accommodating cavity 131, in the film forming stage, the film layer 30 can flow through the pre-coating film layer located in the first accommodating cavity 131 to complement the flow of the film layer 30 adjacent to the first accommodating cavity 131 to ensure that the film Uniform distribution of layers 30 .
  • the film layer 30 When the film layer 30 is located at the second accommodating cavity 132, in the film forming stage, the film layer 30 can pass through the pre-coating film layer located in the second accommodating cavity 132 to complement the flow of the film layer 30 in the adjacent second accommodating cavity 132 to ensure its evenly distributed.
  • the adjacent second accommodating cavities 132 may not communicate with each other. In this case, volatilization or mutual dissolution of the medium outside the film-forming region can be prevented.
  • the film layer 30 when the film layer 30 is located at the second accommodating cavity 132, in the film forming stage, the film layer 30 can pass through the pre-coating film layer located in the second accommodating cavity 132 and the first accommodating cavity 131 in sequence, and flow to the first accommodating cavity 131 The flow complementary to the membrane layer in the adjacent hole unit 13 is achieved.
  • the hole defining layer 10 may include a first defining layer 11 and a second defining layer 12 .
  • the first confinement layer 11 may be disposed on the substrate 20 when assembled with the substrate 20 , and the first accommodating cavity 131 is disposed in the first confinement layer 11 .
  • the first defining layer 11 may include a plurality of first subunits 110 arranged in an array. The first accommodating cavity 131 is disposed in the first subunit 110 .
  • the first confinement layer 11 may include one or more first channels 111 , and the first channels 111 communicate with each adjacent at least two first accommodating cavities 131 .
  • the adjacent can be longitudinally adjacent, laterally adjacent or diagonally adjacent.
  • the first non-polar mediums 31 in the adjacent first accommodating cavities 131 can be complementary in series through the first channels 111 .
  • there are multiple first channels 111 there are many channels through which the first non-polar medium 31 flows, so that the first non-polar medium 31 and/or the film layer 30 can be rapidly connected in series and complementary, and the film formation rate can be improved.
  • the first channel 111 may be a first groove opened in the first confinement layer 11 .
  • the opening direction of the first groove may be a single direction or a plurality of directions.
  • a first slot can be opened along the radial or diagonal direction of the first subunit 110, and the first slot can communicate with two diagonally adjacent first accommodating cavities 131, or even communicate with diagonally adjacent,
  • the four first accommodating cavities 131 adjacent to each other horizontally and vertically realize rapid series complementation of the first non-polar medium 31 , realize series complementation of more channels, and improve the film formation rate.
  • a first groove may be provided along the lateral or longitudinal direction of the first subunit 110, and the first groove may communicate with the first accommodating chambers 131 adjacent to each other in the lateral or longitudinal direction.
  • the first grooves may be formed along the radial direction and the transverse direction of the first subunit 110, respectively, and the first grooves may communicate with the first accommodating chambers 131 adjacent to each other in the transverse direction and the longitudinal direction.
  • the first channel 111 may also be a communicating tube included in the first limiting layer 11 , and the communicating tube may be embedded in the first limiting layer 11 .
  • the specific structural form of the first channel 111 is not limited herein.
  • a groove structure may also be provided on the inner side of the first channel 111 , and the first non-polar medium 31 can occupy a position in the groove structure, which facilitates the series complementation of the first non-polar medium 31 .
  • the flow rate of the first non-polar medium 31 increases, and the speed at which the first non-polar medium 31 and/or the membrane layer 30 complement each other in series can also be increased.
  • the first groove may penetrate through the first confinement layer 11, and the flow cross section of the first groove is relatively large.
  • the first limiting layer 11 may further include at least one first communication groove 112 , the first communication groove 112 communicates with the first accommodating cavity 131 , and the first communication groove 112 extends along the radial direction of the first accommodating cavity 131 . Extending outward, one end of the first communication groove 112 facing away from the first accommodating cavity 131 is closed. At least one first communication groove 112 may form a channel structure extending from the first accommodating cavity 131, and the first communication groove 112 may constitute a capillary structure of the first subunit 110.
  • the first non-polar medium 31 It can stably occupy the space in the capillary structure; and prevent the subsequent first polar medium 32 from entering the first communication groove 112, so as to facilitate the series complementation of the first non-polar medium 31 to the adjacent first accommodating chambers 131, so as to This ensures serial complementarity and stability of the membrane layers 30 .
  • the first communication groove 112 may penetrate through the first limiting layer 11 .
  • the flow cross section of the first communication groove 112 is relatively large, and the first non-polar medium 31 in the first communication groove 112 can be complementary in series in a wider range.
  • the first communication groove 112 may be opened along the radial or diagonal direction of the first subunit 110 .
  • a plurality of the first communication grooves 112 may be evenly formed along the circumferential direction of the first subunit 110 .
  • the plurality of first communication grooves 112 form a plurality of capillary structures to increase the channels through which the first non-polar medium 31 flows.
  • the second confinement layer 12 when the hole array layer structure 10 and the substrate 20 are assembled into one body, the second confinement layer 12 may be located on the side of the first confinement layer 11 away from the substrate 20 , and the second accommodating cavity 132 is disposed on the second confinement layer 11 . Define layer 12 .
  • the second confinement layer 12 may serve as an auxiliary structure for stabilizing the film conformation.
  • the second defining layer 12 may be an interconnected structure or an independent structure.
  • the second limiting layer 12 can play a supporting role for the film layer 30 .
  • the second limiting layer 12 may be an array of third subunits 121 such as a cover plate, the cover plate is an interconnect structure, and each third subunit 121 such as a cover plate may have The third sub-accommodating cavity 1211 .
  • the interconnected cover plates constitute the second limiting layer 12 .
  • the second limiting layer 12 of the interconnect structure can prevent the mutual dissolution or volatilization of the first polar medium 32 and the first non-polar medium 31 in the outer region of the hole unit 13, which is beneficial to the non-polar medium and the polar medium in the non-polar medium.
  • the relative separation of membrane positions has the advantage of storage timeliness.
  • the third subunits 121 distributed in an array may be of an integrated structure, which is convenient for processing and manufacturing.
  • the third sub-accommodating cavity 1211 may be equivalent to the second accommodating cavity 132
  • the hole unit 13 is formed with the first accommodating cavity 131 .
  • the second confinement layer 12 may include a plurality of second subunits 122 distributed in an array, such as pillars, with gaps between adjacent pillars, and each pillar is independent There is a second sub-accommodating cavity 1221 in each column.
  • the second limiting layer 12 is an independent structure.
  • the shape of the cylinder can be a cylinder, a polygonal cylinder, etc., which is not limited here.
  • the media between the adjacent cylinders can communicate with each other, which facilitates the mutual connection and complementation of the first non-polar media 31 , and can improve the stability of the membrane layer 30 .
  • the second sub-accommodating cavity 1221 may be equivalent to the second accommodating cavity 132 , and the hole unit 13 is formed with the first accommodating cavity 131 .
  • the second limiting layer 12 may include a plurality of third subunits 121 arranged in an array and a plurality of second subunits 122 arranged in an array.
  • the third sub-unit 121 has a third sub-accommodating cavity 1211 .
  • the second sub-unit 122 has a second sub-accommodating cavity 1221, the second sub-unit 122 is located in the third sub-accommodating cavity 1211, and the second sub-accommodating cavity 1221 can be used as the second accommodating cavity 132 and the first accommodating cavity 131 to form a hole unit 13.
  • the third sub-accommodating cavity 1211 of the third sub-unit 121 provides a placement area for the second sub-accommodating cavity 1221 of the second sub-unit 122 .
  • other regions between the plurality of third sub-units 121 are continuous structures, which can prevent mutual dissolution or volatilization of the polar medium and the non-polar medium in the region outside the hole unit 13 .
  • the third sub-unit 121 includes a cover plate, and the cover plate has a third sub-accommodating cavity 1211;
  • the second sub-unit 121 includes a cylinder, and the second accommodation cavity 132 is arranged in the cylinder, and each cylinder is located in a corresponding No. 1 of the cover plate.
  • the third subunit 121 is a cover plate
  • the second subunit 122 is a cylinder
  • the cylinder is located in the cover plate, and there is a gap between the cylinder and the cover plate, the cylinder
  • the formation of the interface morphology between the first polar medium 32 and the first non-polar medium 31 is more helpful in the process of film formation.
  • the at least one first communication groove 112 constitutes a capillary structure, and the cylinder and capillary structure are more easily formed to contain the first polar medium 32 and the first non-polar medium. Structural model of the interface of the medium 31 .
  • the second confinement layer 12 may include one or more second channels 125 , and the second channels 125 communicate with each adjacent at least two second accommodating cavities 132 .
  • the adjacent can be longitudinally adjacent, laterally adjacent or diagonally adjacent.
  • the first non-polar mediums 31 in the adjacent second accommodating cavities 132 can be complementary in series through the second channels 125 .
  • a groove structure may also be provided on the inner side of the second channel 125 to facilitate the occupying of the first non-polar medium 31 .
  • the specific example and function of the second channel 125 are basically the same as the above-mentioned specific example of the first channel 111 , and details are not repeated here.
  • At least one first groove 123 is formed on the outer side of the second subunit 122 of the second limiting layer 12 , and the first groove 123 can realize the Complementary in series.
  • the first groove 123 may be opened from the outer side of the second sub-unit 122 in a radial or diagonal direction.
  • the second subunit 122 includes pillars, a plurality of pillars distributed in an array constitute the second limiting layer 12 , and at least one first groove 123 is defined on the outer side of each pillar.
  • Each of the first grooves 123 can be used for the space of the first non-polar medium 31 , so that the first non-polar medium 31 can be complementary in series between adjacent pillars.
  • the second confinement layer 12 may further include at least one second communication groove 124 , the second communication groove 124 communicates with the second accommodating cavity 132 , and the second communication groove 124 extends along the radial direction of the second accommodating cavity 132 . Extending outward, one end of the second communication groove 124 facing away from the second accommodating cavity 132 is closed. At least one second communication groove 124 may form a channel structure extending from the second accommodating cavity 132. During the pre-oiling process, the first non-polar medium 31 may be performed in the second communication groove 124 prior to the first polar medium 32.
  • the second limiting layer 12 includes a plurality of pillars distributed in an array, and at least one second communication groove 124 is defined in the inner side of each pillar.
  • Each column constitutes the second subunit 122 .
  • Each of the second communication grooves 124 can be used to occupy the space of the first non-polar medium 31 , so that the first non-polar medium 31 can be complementary in series between adjacent columns.
  • the second limiting layer 12 includes a plurality of cover plates distributed in an array, each cover plate constitutes a third subunit 121 , and at least one second communication groove 124 is defined in the cover plate .
  • Each of the second communication grooves 124 can be used for occupying the space of the first non-polar medium 31 , so that the first non-polar medium 31 can be complementary in series between adjacent second accommodating cavities 132 .
  • the second defining layer 12 includes a plurality of cover plates distributed in an array and a plurality of pillars distributed in an array.
  • Each cover plate constitutes a third subunit 121
  • each column constitutes a second subunit 122 .
  • the inner side of at least one of the cover plate and the cylinder is provided with at least one second communication groove 124;
  • the inner side and the inner side of the cylinder are both provided with second communication grooves 124 .
  • the specific function of the second communication groove 124 is the same as the above example, and is not repeated here.
  • the diameter or width of the first accommodating cavity 131 is smaller than or equal to the diameter or width of the second accommodating cavity 132 .
  • the second confinement layer 12 can provide the first confinement layer 11 with a stable film. auxiliary structure.
  • At least one first communication groove 112 included in the first limiting layer 11 constitutes a third accommodating cavity 133 , and the diameter or width of the third accommodating cavity 133 may be larger than that of the second accommodating cavity 132 .
  • the diameter or width of the second accommodating cavity 132 may be reduced to be equal to the diameter or width of the first accommodating cavity 131 .
  • the opening of the second accommodating cavity 132 is small, which is not conducive to the sample to be tested entering the hole unit 13 for detection.
  • the second confinement layer 12 can still be the first confinement layer 11 Provides an auxiliary structure to stabilize the membrane.
  • the height of the first accommodating cavity 131 of the first defining layer 11 is greater than the width or diameter of the first accommodating cavity 131 , for example, the height of the first accommodating cavity 131 and the width or diameter of the first accommodating cavity 131
  • the ratio can be 0.8 to 3:1, and the specific ratio can be 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.8:1, 2.0:1, 2.5:1 or 3:1.
  • the specific ratio range can be the ratio range formed by any two of the above numerical values.
  • the first accommodating cavity 131 has a high aspect ratio, which is helpful for the diffusion and occupation of the first non-polar medium 31 and improves the stability of the film formation; and the film layer 30 has a uniform thickness, and the film Layer 30 is functional.
  • At least one of the second subunit 122 and the third subunit 121 and the first subunit 110 may constitute a structural unit of the hole array layer structure 10 , and the width of the structural unit is generally less than 1000 ⁇ m.
  • the hole array layer structure 10 occupies a small space, can include a larger number of structural units, and can detect a larger number of samples.
  • the first communication groove 112 or the second communication groove 112 can form capillary structures of suitable size to facilitate the occupying of the first non-polar medium 31 .
  • FIG. 21 is a schematic structural diagram of a chip device provided according to an embodiment of the present application.
  • the chip device provided in this embodiment of the present application may include a substrate 20 , a hole array layer structure 10 and a film layer 30 according to any of the foregoing embodiments.
  • the hole array layer structure 10 is disposed on the substrate 20; the film layer 30 is located in the plurality of hole units 13 arranged in the array of the hole position defining layer 2, and the film layer 30 is configured to detect the sample to be tested.
  • FIG. 22 shows a schematic diagram of the electrical characterization test.
  • the chip device is electrically characterized by using the electrical characterization device test.
  • the electrical characterization device may be composed of a current detection circuit 42 with a multi-channel adjustable bias voltage.
  • the current detection circuit 42 is electrically connected to the measured object such as the chip device 41 for detecting the performance of the chip device 41 .
  • the current detection circuit 42 may be a transimpedance amplifier (TIA), a capacitive transimpedance amplifier (CTIA) or other forms. Taking a transimpedance amplifier as an example, the electrical characterization works as follows:
  • a bias voltage Vb is applied to the non-inverting input (+) of the operational amplifier. Due to the existence of the negative feedback circuit, the operational amplifier will adjust the output voltage Vo, so that the voltage of its inverting input (-) is the same as that of the non-inverting input, that is, the bias voltage Vb is applied to the measured object.
  • the current flowing on the measured object will all pass through the feedback resistor of the transimpedance amplifier, and the output terminal of the operational amplifier will reflect the current flowing on the measured object in the form of voltage.
  • the feedback resistance is Rf
  • the current I (Vo-Vb)/Rf on the measured object.
  • the DC resistance of the measured object can be obtained by dividing the bias voltage Vb by the current I measured by the current measuring circuit.
  • the color depth of the electrical representation of each unit in the instrument is positively correlated with the value of the membrane capacitance, that is, the darker the color, the greater the membrane capacitance value.
  • it is less than 20pf, it is the background capacitance value of the instrument or the capacitance value of the initial state without film formation, which is displayed in light gray; 20.1 ⁇ 30pf is the membrane capacitance value that is not conducive to the subsequent conventional embedding of the film layer containing amphiphilic material, and it is displayed in gray.
  • 30.1 ⁇ 65pf is the membrane capacitance value suitable for the subsequent conventional embedding of the membrane layer containing amphiphilic molecular materials, which is displayed in dark gray; greater than 65pf is the membrane rupture or the membrane layer containing amphiphilic molecular materials does not have the ability to embed holes. film, shown in black.
  • Figure 23 shows an electrical characterization diagram of the chip device background prior to film formation. As shown in FIG. 23, since the first non-polar medium 31 of the first confinement layer 11 and the second non-polar medium 33 of the second confinement layer 12 are isolated by the second polar medium 32 in the middle, this When the capacitance is only shown as the background value of the circuit system of the instrument itself.
  • FIG. 24 shows an electrical characterization diagram of a chip device as a comparative example.
  • the adjacent first accommodating cavities 131 are connected, and the adjacent second accommodating cavities 131 of the chip device are not connected.
  • the capacitance value after film formation gradually became larger than that before film formation, and the capacitance value at 95% was 45 to 55 pf.
  • FIG. 25 shows an electrical characterization diagram of a chip device according to an embodiment of the present application. As shown in FIG. 25 , the thickness of the film layer 30 is uniform, and the film layer 30 is relatively thin.
  • the series complementation and uniform distribution of the first non-polar medium 31 between different first accommodating cavities 131 can be realized, and the stability of film formation can be improved.
  • an embodiment of the present application provides a film forming method, including steps S100 to S500.
  • FIG. 26 shows a schematic diagram of a film forming process provided by an embodiment of the present application.
  • a chip device is provided, and the chip device includes the hole array layer structure 10 .
  • the chip device may use the chip device of the embodiment of the second aspect of the present application;
  • step S200 the first non-polar medium 31 is arranged in the hole array layer structure 10 , and a precoat film layer is formed on the surface of the hole array layer structure 10 .
  • the hole unit 13 is pre-oiled with the first non-polar medium 31 .
  • the first non-polar medium 31 can be coated on the surface of the hole unit 13 and occupy the surface of the hole unit 13 ; in the subsequent driving step, the first non-polar medium 31 still occupies the surface position of the hole unit 13 .
  • the first non-polar media 31 in different first accommodating cavities 131 can circulate and complement each other, and the first non-polar media 31 can be uniformly distributed; and when the film layer 30 is formed, the film layer 30 can pass through the pre-coating film layer. The complementarity of the adjacent hole units 13 is realized, and the stability of the membrane layer 30 is ensured.
  • Step S300 flowing the first polar medium 32 through the hole array layer structure 10 to replace at least part of the first non-polar medium 31 .
  • the first polar medium 32 partially drives the first non-polar medium 31 .
  • Step S400 flowing the second non-polar medium 33 through the hole array layer structure 10 to replace at least part of the first polar medium 32, wherein the second non-polar medium 33 contains amphiphilic molecular materials.
  • the first polar medium 32 in the hole unit 13 is partially driven out by the second non-polar medium 33 .
  • first non-polar medium 31 and the second non-polar medium 33 can be made of different kinds of substances, and of course the same kind of substances can be selected, but the second non-polar medium 33 may contain amphiphilic molecular materials.
  • the amphiphilic molecular material is dissolved in the second non-polar medium 33, wherein the amphiphilic molecular material refers to a material having both hydrophilicity and lipophilicity.
  • the amphiphilic molecular material can be phospholipids, block Copolymers, etc.
  • 1,2-diphytanoyl-sn-glycero-3-phosphocholine DPhPC
  • distearoyl phosphoethanolamine DSPE
  • diblock copolymers DBCP
  • Tri-block copolymer Tri-block Copolymer
  • Step S500 flowing the second polar medium 34 through the hole array layer structure 10, replacing at least part of the second non-polar medium 33, and forming at the interface between the first polar medium 32 and the second polar medium 34
  • the membrane layer 30, wherein the membrane layer 30 comprises an amphiphilic molecular material may be selected from different types of substances, and of course, the same type of substances may also be selected, which is not specifically limited herein.
  • FIG. 27a is a schematic diagram of film formation as a comparative example
  • FIG. 27b is a schematic diagram of film formation of an embodiment of the present application.
  • step S200 is not used, that is, there is no pre-coating and film-forming process, and the first polar medium 32 is directly filled in the hole array layer structure 10 .
  • the material of the hole array layer structure 10 can be selected from a non-polar material, which has lipophilic and hydrophobic properties, its hydrophobic effect is not enough, for example, the contact angle is less than 120°. If step S200 is removed, and there is no preferential occupation of the first non-polar medium 31, the first polar medium 32 is not hydrophobic enough, and the convex curvature of the liquid surface toward the second non-polar medium 33 is small, and the formed film layer The area of the film 30 is relatively large, the thickness is relatively thick, and the thickness is not uniform, which may easily lead to the loss of functionality of the film layer 30 .
  • the first non-polar medium 31 in the embodiment of the present application is pre-coated in the hole array layer structure 10 , and occupies space in the hole array layer structure 10 prior to the first polar medium 32 .
  • the relative insulation between the hole units 13 avoids crosstalk of electrical signals.
  • the preferential occupation of the first non-polar medium 31 helps the liquid surface of the first polar medium 32 to face the second non-polar medium 33 to have a large convex curvature, and the thickness of the formed film layer 30 is uniform and stable, and the thickness is relatively high. Thin.
  • the first non-polar medium 31 forms a pre-coat film layer on the inner surface of the hole array layer structure, which can guide the flow of the film layer 30 in the adjacent hole units 13 and ensure the film layer 30 stability.
  • an embodiment of the present application provides a nanopore sequencing device, including the hole array layer structure 10 of the first aspect of the present application, the chip device of the second aspect of the present application, or the third aspect of the present application.
  • the film layer 30 prepared by the film forming method.
  • the fifth aspect of the present application relates to the hole array layer structure 10 of the embodiment of the first aspect of the present application, the chip device of the embodiment of the second aspect of the present application, or the film layer 30 prepared by the film-forming method of the embodiment of the third aspect of the present application.
  • the analyte comprises: a biopolymer selected from one of polynucleotides, polypeptides, polysaccharides and lipids.
  • An embodiment of the fifth aspect of the present application provides a method for characterizing an analyte, including using the hole array layer structure 10 of the first aspect of the present application, the chip device of the second aspect of the present application, or the third aspect of the present application
  • the membrane layer 30 prepared by the membrane formation method of the embodiment performs sequencing on the analyte, wherein the analyte includes a biopolymer, and the biopolymer is selected from at least one of polynucleotides, polypeptides, polysaccharides and lipids. Examples of biopolymers may be polynucleotides, including DNA and/or RNA.

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Abstract

本申请公开了孔阵列层结构、芯片装置、纳米孔测序装置和成膜方法以及用途。孔阵列层结构，用于和衬底形成成膜空间，成膜空间用于形成膜层。孔阵列层结构包括：阵列排布的多个孔单元，孔单元贯穿孔阵列层结构，孔单元具有轴向连通的第一容纳腔和第二容纳腔，第一容纳腔被配置为和衬底连接，至少一个第一容纳腔与其它第一容纳腔连通。本申请提供的孔阵列层结构能够实现第一非极性介质在不同第一容纳腔之间的串联互补，以及均匀性分布，可以保证预涂的均一性，为成膜提供较佳的成膜基础；并且在成膜阶段可以保证膜层的稳定性和串联互补。

Description

孔阵列层结构、芯片装置、纳米孔测序装置和成膜方法以及用途 技术领域
本申请涉及生物检测技术领域，特别是涉及孔阵列层结构、芯片装置、纳米孔测序装置和成膜方法以及用途。
背景技术
纳米孔检测技术，具有高通量、高集成、平行化、多样化和自动化等优点，在蛋白质检测、基因测序和纳米颗粒的表征等领域具有广泛的应用。例如当进行基因测序时，基因测序装置的薄膜中设置有纳米孔，单链脱氧核糖核酸(DeoxyriboNucleic Acid，DNA)分子穿过纳米孔时，由于不同碱基的形状大小有差异，与纳米孔内检测分子发生特异性反应从而引起电阻变化。纳米孔的两侧具有恒定电压，可以检测到不同碱基经过纳米孔中电流的变化，从而反映出通过纳米孔的DNA分子的碱基排列情况。
基因测序装置的薄膜的稳定性对检测的准确性至关重要，但是目前薄膜的稳定性不佳，从而导致检测的准确性较差。
发明内容
本申请提供孔阵列层结构、芯片装置、纳米孔测序装置和成膜方法以及用途，旨在解决薄膜稳定性不佳的问题，可以提高成膜的稳定性。
第一方面，本申请实施例提供了一种孔阵列层结构，用于和衬底形成成膜空间，成膜空间用于形成膜层。孔阵列层结构包括：阵列排布的多个孔单元，孔单元贯穿孔阵列层结构，孔单元具有轴向连通的第一容纳腔和第二容纳腔，第一容纳腔被配置为和衬底连接，至少一个第一容纳腔与其它第一容纳腔连通。
根据本申请第一方面的实施例，至少一个第二容纳腔与其它第二容纳腔连通。
根据本申请第一方面的前述任一实施例，孔阵列层结构包括第一限定层和第二限定层，第一容纳腔设置于第一限定层内并贯通第一限定层，第二容纳腔设置于第二限定层内并贯通第二限定层。
根据本申请第一方面的前述任一实施例，第二限定层包括阵列排布的多个第二子单元，第二容纳腔设置于第二子单元内并贯通第二子单元。
根据本申请第一方面的前述任一实施例，第二限定层包括阵列排布的多个第三子单元，第三子单元包括第三子容纳腔，第二子单元位于第三子容纳腔内，第二子单元和第三子单元之间具有间隙。
根据本申请第一方面的前述任一实施例，第二子单元的外侧开设有多个第一凹槽。
根据本申请第一方面的前述任一实施例，第一限定层包括至少一个第一通道，第一通道将每相邻的至少两个第一容纳腔连通。
根据本申请第一方面的前述任一实施例，第二限定层包括至少一个第二通道，第二通道将每相邻的至少两个第二容纳腔连通。
根据本申请第一方面的前述任一实施例，第一限定层包括至少一个第一连通槽，第一连通槽沿第一容纳腔的径向向外延伸，第一连通槽背离第一容纳腔的一端封闭。
根据本申请第一方面的前述任一实施例，第一限定层包括至少一个第一连通槽，第一连通槽贯通第一限定层。
根据本申请第一方面的前述任一实施例，第二限定层包括至少一个第二连通槽，第二连通槽沿第二容纳腔的径向向外延伸，第二连通槽背离第二容纳腔的一端封闭。
根据本申请第一方面的前述任一实施例，第二限定层包括至少一个第二连通槽，第二连通槽贯通第二限定层。
根据本申请第一方面的前述任一实施例，第一限定层包括至少一个第一连通槽，至少一个第一连通槽形成第三容纳腔，第三容纳腔的直径或宽度大于第二容纳腔的直径或宽度。
根据本申请第一方面的前述任一实施例，第一容纳腔的直径或宽度小于或等于第二容纳腔的直径或宽度。
第二方面，本申请实施例提供了一种芯片装置，其包括如衬底；上述第一方面实施例的孔阵列层结构，孔阵列层结构位于衬底上，孔阵列层结构包括阵列排布的多个孔单元，孔单元的第二容纳腔位于孔单元的第一容纳腔背离衬底的一侧；以及膜层，膜层位于多个孔单元内，并且膜层被配置为检测待测样本。
第三方面，本申请实施例提供了一种成膜方法，包括：将第一非极性介质布置于孔阵列层结构内，于孔阵列层结构的表面形成预涂膜层；将第一极性介质流动穿过孔阵列层结构内，至少取代部分第一非极性介质；将第二非极性介质流动穿过孔阵列层结构，至少取代部分第一极性介质，其中，第二非极性介质包含两亲性分子材料；将第二极性介质流动穿过孔阵列层结构，至少取代部分第一非极性介质，并在第一极性介质和第二极性介质之间的界面形成膜层，其中，膜层包含两亲性分子材料。
第四方面，本申请实施例提供了一种纳米孔测序装置，包括本申请第一方面实施例的孔阵列层结构、本申请第二方面实施例的芯片装置或者本申请第三方面实施例的成膜方法制备的膜层。
第五方面，本申请第一方面实施例的孔阵列层结构、本申请第二方面实施例的芯片装置或者本申请第三方面实施例的成膜方法制备的膜层在表征分析物中的用途，分析物包括：生物聚合物，生物聚合物选自多核苷酸、多肽、多糖和脂质中的一种。
根据本申请第五方面的前述任一实施例，生物聚合物为多核苷酸，多核苷酸包括 DNA和/或RNA。
根据本申请实施例的孔阵列层结构，孔阵列层结构包括阵列排布的多个孔单元，于孔单元内形成膜层；每个孔单元包括彼此连通的第一容纳腔和第二容纳腔，至少一个第一容纳腔与其它第一容纳腔连通，位于第一容纳腔内的第一非极性介质能够在相邻的第一容纳腔扩散流动，实现第一非极性介质在不同第一容纳腔之间的串联互补，以及均匀性分布；一方面可以保证预涂的均一性，为成膜提供较佳的成膜基础；另一方面，在成膜阶段可以保证膜层的稳定性和串联互补。
附图说明
下面将参考附图来描述本申请示例性实施例的特征、优点和技术效果。
图1是根据本申请一种实施例提供的孔阵列层结构的立体图；
图2是成膜示意图；
图3是根据本申请一种实施例提供的孔位限定层的立体分解图；
图4是根据本申请一种实施例提供的孔位限定层的俯视图；
图5是根据本申请第一种替代实施例提供的孔位限定层的立体分解图；
图6是根据本申请第一种替代实施例提供的孔位限定层的俯视图；
图7是根据本申请第一种替代实施例提供的第二限定层的俯视图；
图8是根据本申请第二种替代实施例提供的孔位限定层的立体图；
图9是根据本申请第二种替代实施例提供的孔位限定层的立体分解图；
图10是根据本申请第二种替代实施例提供的孔位限定层的俯视图；
图11是根据本申请第三种替代实施例提供的孔位限定层的立体分解图；
图12是根据本申请第三种替代实施例提供的孔位限定层的俯视图；
图13是根据本申请第三种替代实施例提供的第二限定层的俯视图之一；
图14是根据本申请第三种替代实施例提供的第二限定层的俯视图之二；
图15是根据本申请第三种替代实施例提供的第二限定层的俯视图之三；
图16是根据本申请第四种替代实施例提供的孔位限定层的立体分解图；
图17是根据本申请第四种替代实施例提供的孔位限定层的俯视图；
图18是根据本申请第四种替代实施例提供的第二限定层的俯视图；
图19是图18的A-A剖面示意图；
图20是根据本申请第五种替代实施例提供的第二限定层的俯视图；
图21是根据本申请一种实施例提供的芯片装置的结构示意图；
图22示出了电学表征测试原理图；
图23是根据本申请一种实施例提供的未成膜之前的芯片装置本底的电学表征图；
图24是根据对比示例的芯片装置的电学表征图；
图25是根据本申请一种实施例提供的芯片装置的电学表征图；
图26是根据本申请一种实施例提供的成膜流程示意图；
图27是膜层结构示意图。
具体实施方式
下面结合附图和实施例对本申请的实施方式作进一步详细描述。以下实施例的详细描述和附图用于示例性地说明本申请的原理，但不能用来限制本申请的范围，即本申请不限于所描述的实施例。
为了更好地理解本申请，下面结合图1～图27对本申请实施例进行描述。第一方面，本申请实施例提供了一种孔阵列层结构。图1是根据本申请实施例提供的孔阵列层结构的结构示意图。图2是根据本申请实施例提供的成膜示意图。如图1和图2所示，本申请实施例提供的孔阵列层结构10，可以用于和衬底20形成成膜空间，成膜空间用于形成膜层30。孔阵列层结构10包括阵列排布的多个孔单元13，每个孔单元13贯穿孔阵列层结构10。每个孔单元13包括轴向连通的第一容纳腔131和第二容纳腔132。第一容纳腔131被配置为和衬底20连接。第一容纳腔131贯穿孔阵列层结构10靠近衬底20的一侧，第二容纳腔132贯穿孔阵列层结构10背离衬底20的一侧，至少一个第一容纳腔131与其它第一容纳腔131连通。
如图2所示，根据本申请实施例的孔阵列层结构10，膜层30形成于孔单元13内。具体地，成膜步骤可以包括步骤S200、步骤300、步骤400和步骤500。
在步骤S200中，将第一非极性介质31布置于孔阵列层结构10内，于孔阵列层结构10的表面形成预涂膜层。
在步骤S300中，将第一极性介质32流动穿过孔阵列层结构10内，至少取代部分第一非极性介质31。
在步骤S400中，将第二非极性介质33流动穿过孔阵列层结构10，至少取代部分第一极性介质32，其中，第二非极性介质33包含两亲性分子材料。
在步骤S500中，将第二极性介质34流动穿过孔阵列层结构10，至少取代部分第二非极性介质33，并在第一极性介质32和第二极性介质34之间的界面形成膜层30，膜层30包含两亲性分子材料。
根据本申请实施例的孔阵列层结构10，孔阵列层结构10包括阵列排布的多个孔单元13，于孔单元13内形成膜层30；每个孔单元13包括彼此连通的第一容纳腔131和第二容纳腔132，至少一个第一容纳腔131与其它第一容纳腔131连通，位于第一容纳腔131内的第一非极性介质31能够在相邻的第一容纳腔131扩散流动，并在孔单元13的表面形成预涂膜层，实现第一非极性介质31在不同第一容纳腔131之间的串联互 补，以及均匀性分布；一方面可以保证预涂的均一性，为成膜提供较佳的成膜基础。另一方面，膜层30的溶剂为第二非极性介质33，第二非极性介质33和第一非极性介质31相容性较好，因此膜层30和预涂膜层的相容性较好；在成膜阶段，膜层30可以通过预涂膜层与相邻第一容纳腔131的膜层30流动互补，可以保证膜层30的稳定性和串联互补。
如图2所示，在一些实施例中，至少一个第二容纳腔132与其它第二容纳腔132连通。第一非极性介质31能够在相邻的第二容纳腔132之间扩散流动，实现第一非极性介质31在不同第二容纳腔132之间的串联互补，进一步保证膜层30的稳定性和串联互补。
当膜层30位于第一容纳腔131处，成膜阶段，膜层30可以通过位于第一容纳腔131的预涂膜层，与相邻第一容纳腔131的膜层30流动互补，保证膜层30的均匀分布。当膜层30位于第二容纳腔132处，成膜阶段，膜层30可以通过位于第二容纳腔132的预涂膜层，与相邻第二容纳腔132的膜层30的流动互补，保证其均匀分布。
当然，相邻第二容纳腔132之间也可以不连通。在此情况下，可以防止成膜区域外的介质挥发或互溶等。并且，当膜层30位于第二容纳腔132处，在成膜阶段，膜层30可以依次通过位于第二容纳腔132、第一容纳腔131的预涂膜层，流至第一容纳腔131内，实现与相邻孔单元13内的膜层的流动互补。
如图3所示，在一些实施例中，孔位限定层10可以包括第一限定层11和第二限定层12。第一限定层11，在和衬底20组装为一体时，可以设置于衬底20上，第一容纳腔131设置于第一限定层11。第一限定层11可以包括阵列排布的多个第一子单元110。第一容纳腔131设置于第一子单元110中。
如图3所示，在一些实施例中，第一限定层11可以包括一个或多个第一通道111，第一通道111将每相邻的至少两个第一容纳腔131连通。其中，相邻可以是纵向相邻、横向相邻或对角线相邻。相邻的第一容纳腔131内的第一非极性介质31可以通过第一通道111实现串联互补。当第一通道111为多个时，第一非极性介质31流通的通道较多，可实现第一非极性介质31和/或膜层30的快速串联互补，提高成膜速率。
作为示例，第一通道111可以为第一限定层11开设的第一槽。第一槽的开设方向可以为单一方向，也可以为多个方向。例如，可以沿第一子单元110的径向或对角线方向开设第一槽，第一槽可以连通对角线相邻的两个第一容纳腔131，甚至可连通对角线相邻、横向相邻和竖向相邻的四个第一容纳腔131，实现第一非极性介质31的快速串联互补，实现更多通道的串联互补，提高成膜速率。又例如，可以沿第一子单元110的横向或纵向开设第一槽，第一槽可以连通横向或纵向相邻的第一容纳腔131。再例如，可以沿第一子单元110的径向和横向分别开设第一槽，第一槽可以连通横向和纵向相邻的第一容纳腔131。
作为另一示例，第一通道111也可以为第一限定层11包括的连通管，连通管可以嵌入第一限定层11中。在此并不对第一通道111的具体结构形式进行限定。
可选地，在第一通道111的内侧也可开设凹槽结构，第一非极性介质31能够在凹槽结构中占位，利于第一非极性介质31的串联互补。
随着第一通道111的流通截面的增大，第一非极性介质31的流通量增加，也可增加第一非极性介质31和/或膜层30串联互补的速度。例如，第一槽可以贯通第一限定层11，第一槽的流通截面较大。
在一些实施例中，第一限定层11还可以包括至少一个第一连通槽112，第一连通槽112与第一容纳腔131连通，第一连通槽112沿第一容纳腔131的径向向外延伸，第一连通槽112背离第一容纳腔131的一端封闭。至少一个第一连通槽112可以形成第一容纳腔131延伸的通道结构，并且第一连通槽112可以构成第一子单元110的毛细管结构，在预涂油过程中，第一非极性介质31可以在毛细管结构内稳定占位；并阻止后续的第一极性介质32进入第一连通槽112内，便于对相邻的第一容纳腔131进行第一非极性介质31的串联互补，以此保证膜层30的串联互补和稳定性。
可选地，第一连通槽112可以贯通第一限定层11。第一连通槽112的流通截面较大，第一连通槽112内的第一非极性介质31可以进行更大范围的串联互补。
作为示例，第一连通槽112可以沿第一子单元110的径向或对角线方向开设。当然第一连通槽112也可以沿第一子单元110周向均匀开设多个。多个第一连通槽112构成多个毛细管结构，增加第一非极性介质31流通的通道。
根据本申请的实施例，将孔阵列层结构10与衬底20组装为一体时，第二限定层12可以位于第一限定层11背离衬底20一侧，第二容纳腔132设置于第二限定层12。第二限定层12可以作为稳定膜构象的辅助结构。
在一些实施例中，第二限定层12可以为互联结构或者独立结构。第二限定层12可以对膜层30起到支撑作用。
如图3～图7所示，作为示例，第二限定层12可以为阵列分布的第三子单元121例如盖板，盖板为互联结构，每个第三子单元121如盖板均可以具有第三子容纳腔1211。互联的盖板构成第二限定层12。互联结构的第二限定层12可以防止孔单元13外区域的第一极性介质32和第一非极性介质31的互溶或挥发等情况，有利于非极性介质和极性介质在非成膜位置的相对分离，具有储存时效性的优势。其中，阵列分布的第三子单元121可以为一体式结构，方便加工制造。此时，第三子容纳腔1211可以相当于第二容纳腔132，和第一容纳腔131构成孔单元13。
如图8～图15所示，作为另一示例，第二限定层12可以包括阵列分布的多个第二子单元122例如柱体，相邻柱体之间具有间隙，每个柱体为独立结构，每个柱体内具有第二子容纳腔1221。第二限定层12为独立结构。柱体的形状可以为圆柱、多边柱等，在此不对其进行限定。相邻柱体之间介质可以互通，利于第一非极性介质31的相互串联和互补，可以提高膜层30的稳定性。此时，第二子容纳腔1221可以相当于第二容纳腔132，和第一容纳腔131构成孔单元13。
如图16～图19所示，作为再一示例，第二限定层12可以包括阵列排布的多个第三子单元121和阵列排布的多个第二子单元122。其中，第三子单元121具有第三子容纳腔1211。第二子单元122具有第二子容纳腔1221，第二子单元122位于第三子容纳腔1211内，并且第二子容纳腔1221可以作为第二容纳腔132与第一容纳腔131构成孔单元13。第三子单元121的第三子容纳腔1211为第二子单元122的第二子容纳腔1221提供放置区域。并且多个第三子单元121之间的其他区域为连续结构，能够防止孔单元13外区域的极性介质和非极性介质的互溶或挥发等情况。例如，第三子单元121包括盖板，盖板具有第三子容纳腔1211；第二子单元121包括柱体，第二容纳腔132设置于柱体内，每个柱体位于盖板相应的第三子容纳腔1211内。
根据本申请的实施例，第三子单元121和第二子单元122之间可以具有空隙，有利用控制成膜过程中膜层30的形貌。
如图16～图19所示，作为示例，第三子单元121为盖板，第二子单元122为圆柱体，圆柱体位于盖板内，并且圆柱体和盖板之间具有间隙，圆柱体在成膜的过程中更有助于第一极性介质32和第一非极性介质31的界面形貌的形成。
尤其是，第一限定层11包括至少一个第一连通槽112时，至少一个第一连通槽112构成毛细管结构，圆柱体和毛细管结构更容易形成包含第一极性介质32和第一非极性介质31的分界面的结构模型。
如图20所示，在一些实施例中，第二限定层12可以包括一个或多个第二通道125，第二通道125将每相邻的至少两个第二容纳腔132连通。其中，相邻可以是纵向相邻、横向相邻或对角线相邻。相邻的第二容纳腔132内的第一非极性介质31可以通过第二通道125实现串联互补。
可选地，第二通道125的内侧还可以设置凹槽结构，便于第一非极性介质31的占位。需要说明的是，第二通道125的具体示例和作用与第一通道111的上述具体示例基本相同，在此不再赘述。
在一些实施例中，第二限定层12的第二子单元122的外侧开设有至少一个第一凹槽123，第一凹槽123能够实现第一非极性介质31在第二限定层12的串联互补。
如图11～14所示，作为示例，第一凹槽123可以为第二子单元122由其外侧沿径向或对角线方向开设。例如，第二子单元122包括柱体，阵列分布的多个柱体构成第二限定层12，每个柱体的外侧开设至少一个第一凹槽123。每个第一凹槽123可以用于第一非极性介质31的占位，便于第一非极性介质31在相邻柱体之间进行串联互补。
在一些实施例中，第二限定层12还可以包括至少一个第二连通槽124，第二连通槽124与第二容纳腔132连通，第二连通槽124沿第二容纳腔132的径向向外延伸，第二连通槽124背离第二容纳腔132的一端封闭。至少一个第二连通槽124可以形成第二容纳腔132延伸的通道结构，在预涂油过程中，第一非极性介质31可以优先于第一极性介质32在第二连通槽124内进行占位，并阻止第一极性介质32进入第二连通 槽124内，便于对相邻的第二容纳腔132进行第一非极性介质31的串联互补，以此保证膜层30的串联互补和稳定性。需要说明的是，第二连通槽124的具体示例和作用与第一连通槽112的具体示例和作用相同，在此不再赘述。
如图13所示，作为示例，第二限定层12包括阵列分布的多个柱体，每个柱体的内侧开设至少一个第二连通槽124。每个柱体构成第二子单元122。每个第二连通槽124可以用于第一非极性介质31的占位，便于第一非极性介质31在相邻柱体之间进行串联互补。
如图5～7所示，作为另一示例，第二限定层12包括阵列分布的多个盖板，每个盖板构成一个第三子单元121，盖板内开设至少一个第二连通槽124。每个第二连通槽124可以用于第一非极性介质31的占位，便于第一非极性介质31在相邻第二容纳腔132之间进行串联互补。
作为再一示例，第二限定层12包括阵列分布的多个盖板和阵列分布的多个柱体。每个盖板构成一个第三子单元121，每个柱体构成一个第二子单元122。盖板和柱体中的至少一种的内侧开设有至少一个第二连通槽124；例如盖板内侧开设第二连通槽124，又例如柱体内侧开设第二连通槽124，再例如，盖板内侧和柱体内侧均开设第二连通槽124。第二连通槽124的具体作用和上述示例一致，在此不再赘述。
根据本申请的实施例，第一容纳腔131的直径或宽度小于或等于第二容纳腔132的直径或宽度，在此状态下，第二限定层12能够为第一限定层11提供稳定膜的辅助结构。
在一些实施例中，第一限定层11包括的至少一个第一连通槽112构成第三容纳腔133，第三容纳腔133的直径或宽度可以大于第二容纳腔132的直径或宽度。
以第一容纳腔131的直径或宽度为基准，在第二容纳腔132的直径或宽度大于第一容纳腔131的直径或宽度时，随着第二容纳腔132的直径或宽度的减小，更有利于形成尺寸较小的膜层30，膜层30的状态更加稳定。第二容纳腔132的直径或宽度可以减小至等于第一容纳腔131的直径或宽度。但是当继续减小第二容纳腔132的直径或宽度时，第二容纳腔132的开窗较小，不利于待测样本进入孔单元13中进行检测。
在第二容纳腔132的直径或宽度小于或等于第三容纳腔133的直径或宽度，随着第二容纳腔132的直径或宽度的增大，第二限定层12仍然能够为第一限定层11提供稳定膜的辅助结构。
根据本申请实施例的第一限定层11的第一容纳腔131的高度大于第一容纳腔131的宽度或直径，例如，第一容纳腔131的高度与第一容纳腔131的宽度或直径的比例可以为0.8～3：1，其比例具体可以为0.8：1，0.9：1，1.0：1，1.1：1，1.2：1，1.3：1，1.4：1、1.5：1、1.8：1、2.0：1、2.5：1或3：1。具体比例范围可以为以上任意两个数值构成的比例范围。在上述数据范围内，第一容纳腔131具有较高的深宽比，有助于第一非极性介质31的扩散和占位，提高成膜的稳定性；且膜层30厚度均匀，膜层30功能性较佳。
根据本申请实施例的第二子单元122和第三子单元121中的至少一个、以及第一子单元110可以构成孔阵列层结构10的一个结构单元，该结构单元的宽度一般为1000μm以下。孔阵列层结构10占用空间小，且可包含较多数量的结构单元，可检测更多数量的样本。
第一容纳腔131的高度和第一连通槽112的宽度比值为3以上，或者第二容纳腔132的高度和第二连通槽124的宽度比值为3以上，第一连通槽112或第二连通槽124可以形成尺寸合适的毛细管结构，便于第一非极性介质31的占位。
第二方面，本申请实施例提供了一种芯片装置。图21是根据本申请实施例提供的芯片装置的结构示意图。如图21所示，本申请实施例提供的芯片装置可以包括衬底20、上述任一实施例的孔阵列层结构10和膜层30。孔阵列层结构10设置于衬底20上；膜层30位于孔位限定层2的阵列排布的多个孔单元13内，并且膜层30被配置为检测待测样本。
图22示出了电学表征测试原理图，如图22所示，采用电学表征装置测试对芯片装置进行电学表征。电学表征装置可以由多通道可调节偏置电压的电流检测电路42构成，电流检测电路42和被测对象例如芯片装置41电连接，用于检测芯片装置41的性能。其中，电流检测电路42可以是跨阻放大器(TIA)、电容跨阻放大器(CTIA)或其它形式。以跨阻放大器为例，电学表征的工作原理如下：
在运算放大器的同相输入端(+)施加偏置电压Vb。由于负反馈电路的存在，运算放大器会调整输出电压Vo，从而使其反向输入端(-)的电压与同相输入端相同，也就是将偏置电压Vb施加在被测对象上。
由于运算放大器输入端阻抗非常大，可认为没有电流流入或流出。所以被测对象上流过的电流，会全部通过跨阻放大器的反馈电阻，在运算放大器的输出端以电压的形式反应被测对象上流过的电流。若反馈电阻为Rf，则被测对象上的电流I＝(Vo-Vb)/Rf。用偏置电压Vb除以电流测量电路测得的电流I即可得到被测对象的直流电阻。检测被测对象的电容，只需将特定的交流信号(如正弦波、三角波等)施加在被测对象上，检测其电流响应，从而计算出被测对象的电容。
其中，仪器中每个单元电学表征的显示颜色深浅与膜电容值的大小成正相关，即颜色越深代表膜电容值越大。当小于20pf为仪器本底电容值或未成膜初始状态的电容值，显示为浅灰色；20.1～30pf为不利于包含两亲分子材料的膜层进行后续常规嵌孔的膜电容值，显示为灰色；30.1～65pf为适合包含两亲分子材料的膜层进行后续常规嵌孔的膜电容值，显示为深灰色；大于65pf为破膜或包含两亲分子材料的膜层呈现出不具备嵌孔能力的膜，显示为黑色。
图23示出了在未成膜之前的芯片装置本底的电学表征图。如图23所示，由于第一限定层11的第一非极性介质31和第二限定层12的第二非极性介质33被处于二者中间的第二极性介质32隔绝，因此此时电容仅表现为仪器本身电路系统的本底值。
图24示出了作为对比示例的芯片装置的电学表征图。对比示例的芯片装置的相邻第一容纳腔131连通，芯片装置的相邻第二容纳腔131之间不连通。如图24所示，相比未成膜之前，成膜后的电容值逐渐变大，95％的电容值为45～55pf。
图25示出了根据本申请实施例的芯片装置的电学表征图。如图25所示，膜层30厚度均匀，且膜层30较薄。
根据本申请实施例的芯片装置，能够实现第一非极性介质31在不同第一容纳腔131之间的串联互补，以及均匀性分布，可以提高成膜的稳定性。
第三方面，本申请实施例提供了一种成膜方法，包括：步骤S100至步骤S500。
图26示出了本申请一种实施例提供的成膜流程示意图。如图26所示，步骤S100，提供芯片装置，芯片装置包括孔阵列层结构10。其中，芯片装置可以使用本申请第二方面实施例的芯片装置；
步骤S200，将第一非极性介质31布置于孔阵列层结构10内，于孔阵列层结构10的表面形成预涂膜层。采用第一非极性介质31对孔单元13进行预涂油处理。
第一非极性介质31能够涂覆于孔单元13的表面，并占据孔单元13的表面；在后续的驱赶步骤中，第一非极性介质31仍占据孔单元13的表面位置。不同第一容纳腔131内的第一非极性介质31能够互相流通互补，可以将第一非极性介质31均匀性分布；并且在形成膜层30时，膜层30可通过预涂膜层实现相邻孔单元13的互补，保证膜层30的稳定性。
步骤S300，将第一极性介质32流动穿过孔阵列层结构10内，至少取代部分第一非极性介质31。第一极性介质32对第一非极性介质31进行部分驱赶。
步骤S400，将第二非极性介质33流动穿过孔阵列层结构10，至少取代部分第一极性介质32，其中，第二非极性介质33包含两亲性分子材料。利用第二非极性介质33对孔单元13内的第一极性介质32进行部分驱赶。
需要说明的是，第一非极性介质31和第二非极性介质33可以选取不同种类的物质，当然也可选取相同种类的物质，但是第二非极性介质33中可以包含两亲性分子材料。两亲性分子材料溶于第二非极性介质33中，其中两亲性分子材料是指同时具有亲水性以及亲脂性的材料，作为示例，两亲性分子材料可以为磷脂类、嵌段共聚物类等。例如，二植酰磷脂酰胆碱(1,2-diphytanoyl-sn-glycero-3-phosphocholine，DPhPC)、二硬脂酰基磷脂酰乙醇胺(Distearoyl phosphoethanolamine，DSPE)、两嵌段共聚物(Diblock Copolymers，DBCP)、三嵌段共聚物(Tri-block Copolymer，TBCP)等。
步骤S500，将第二极性介质34流动穿过孔阵列层结构10，至少取代部分第二非极性介质33，并在第一极性介质32和第二极性介质34之间的界面形成膜层30，其中，膜层30包含两亲性分子材料。需要说明的是，第一极性介质32和第二极性介质34可以选取不同种类的物质，当然也可以选取相同种类的物质，在此不作具体的限定。
请参阅图27，图27a是作为对比示例的成膜示意图，图27b为本申请实施例的成膜示意图。如图27a所示，对比示例中不采用步骤S200，即没有预涂成膜的过程，直接采用第一极性介质32填充于孔阵列层结构10内。
虽然孔阵列层结构10的材质可选用非极性材料，具有亲油疏水特性，但是其疏水效果不够例如接触角小于120°。如果去掉步骤S200，没有第一非极性介质31的优先占位，则第一极性介质32因不够疏水，朝向第二非极性介质33的液面上凸曲率较小，形成的膜层30的面积较大，厚度较厚，且厚度不均匀，容易导致膜层30丧失功能性。
而本申请实施例的第一非极性介质31预涂于孔阵列层结构10内，优先于第一极性介质32在孔阵列层结构10内占位，可以保证孔阵列层结构10的各个孔单元13之间的相对绝缘性，避免电信号的串扰。并且第一非极性介质31的优先占位，有助于第一极性介质32朝向第二非极性介质33的液面上凸曲率大，形成的膜层30的厚度均匀稳定且厚度较薄。
根据本申请实施例的成膜方法，第一非极性介质31在孔阵列层结构的内表面形成预涂膜层，可以引导膜层30在相邻孔单元13的流动，保证膜层30的稳定性。
第四方面，本申请实施例提供了一种纳米孔测序装置，包括本申请第一方面实施例的孔阵列层结构10、本申请第二方面实施例的芯片装置或者本申请第三方面实施例的成膜方法制备的膜层30。
本申请的第五方面涉及本申请第一方面实施例的孔阵列层结构10、本申请第二方面实施例的芯片装置或者本申请第三方面实施例的成膜方法制备的膜层30在表征分析物中的用途，其中，分析物包括：生物聚合物，生物聚合物选自多核苷酸、多肽、多糖和脂质中的一种。
本申请第五方面的实施例提供了一种表征分析物的方法，包括使用本申请第一方面实施例的孔阵列层结构10、本申请第二方面实施例的芯片装置或者本申请第三方面实施例的成膜方法制备的膜层30对分析物进行测序，其中，分析物包括生物聚合物，生物聚合物选自多核苷酸、多肽、多糖和脂质中的至少一种。生物聚合物的示例可为多核苷酸，多核苷酸包括DNA和/或RNA。
虽然已经参考优选实施例对本申请进行了描述，但在不脱离本申请的范围的情况下，可以对其进行各种改进并且可以用等效物替换其中的部件。尤其是，只要不存在结构冲突，各个实施例中所提到的各项技术特征均可以任意方式组合起来。本申请并不局限于文中公开的特定实施例，而是包括落入权利要求的范围内的所有技术方案。

Claims (12)

  1. 一种孔阵列层结构，用于和衬底形成成膜空间，所述成膜空间用于形成膜层，所述孔阵列层结构包括：阵列排布的多个孔单元，所述孔单元贯穿所述孔阵列层结构，所述孔单元具有轴向连通的第一容纳腔和第二容纳腔，所述第一容纳腔被配置为和所述衬底连接，至少一个所述第一容纳腔与其它所述第一容纳腔连通。
  2. 根据权利要求1所述的孔阵列层结构，其中，至少一个所述第二容纳腔与其它所述第二容纳腔连通。
  3. 根据权利要求1或2所述的孔阵列层结构，其中，所述孔阵列层结构包括第一限定层和第二限定层，所述第一容纳腔设置于所述第一限定层内并贯通所述第一限定层，所述第二容纳腔设置于所述第二限定层内并贯通所述第二限定层。
  4. 根据权利要求3所述的孔阵列层结构，其中，所述第二限定层包括阵列排布的多个第二子单元，所述第二容纳腔设置于所述第二子单元内并贯通所述第二子单元。
  5. 根据权利要求4所述的孔阵列层结构，其中，所述第二限定层还包括阵列排布的多个第三子单元，所述第三子单元包括第三子容纳腔，所述第二子单元位于所述第三子容纳腔内，所述第二子单元和所述第三子单元之间具有间隙；
    可选地，所述第二子单元的外侧开设有多个第一凹槽。
  6. 根据权利要求3所述的孔阵列层结构，其中，所述第一限定层包括至少一个第一通道，所述第一通道将每相邻的至少两个所述第一容纳腔连通；和/或
    所述第二限定层包括至少一个第二通道，所述第二通道将每相邻的至少两个所述第二容纳腔连通。
  7. 根据权利要求3所述的孔阵列结构，其中，所述第一限定层包括至少一个第一连通槽，所述第一连通槽沿所述第一容纳腔的径向向外延伸，所述第一连通槽背离所述第一容纳腔的一端封闭；可选地，所述第一连通槽贯通所述第一限定层；和/或
    所述第二限定层包括至少一个第二连通槽，所述第二连通槽沿所述第二容纳腔的径向向外延伸，所述第二连通槽背离所述第二容纳腔的一端封闭；可选地，所述第二连通槽贯通所述第二限定层。
  8. 根据权利要求7所述的孔阵列结构，其中，所述第一限定层包括至少一个第一连通槽，至少一个所述第一连通槽形成第三容纳腔，所述第三容纳腔的直径或宽度大于所述第二容纳腔的直径或宽度；可选地，所述第一容纳腔的直径或宽度小于或等于 所述第二容纳腔的直径或宽度。
  9. 芯片装置，包括：
    衬底；
    如权利要求1所述的孔阵列层结构，所述孔阵列层结构位于所述衬底上，所述孔阵列层结构包括阵列排布的多个孔单元，所述孔单元的第二容纳腔位于所述孔单元的第一容纳腔背离所述衬底的一侧；以及
    膜层，所述膜层位于所述多个孔单元内，并且所述膜层被配置为检测待测样本。
  10. 一种成膜方法，包括：
    提供如权利要求9所述的芯片装置，所述芯片装置包括孔阵列层结构；
    将第一非极性介质布置于孔阵列层结构内，于所述孔阵列层结构的表面形成预涂膜层；
    将第一极性介质流动穿过所述孔阵列层结构内，至少取代部分所述第一非极性介质；
    将第二非极性介质流动穿过所述孔阵列层结构，至少取代部分所述第一极性介质，其中，所述第二非极性介质包含两亲性分子材料；
    将第二极性介质流动穿过所述孔阵列层结构，至少取代部分所述第二非极性介质，并在所述第一极性介质和所述第二极性介质之间的界面形成膜层，其中，所述膜层包含两亲性分子材料。
  11. 一种纳米孔测序装置，包括权利要求1～8任一项所述的孔阵列层结构、权利要求9所述的芯片装置或权利要求10所述的成膜方法制备的膜层。
  12. 权利要求1～8任一项所述的孔阵列层结构、权利要求9所述芯片装置或权利要求10所述的成膜方法制备的膜层在表征分析物中的用途，所述分析物包括：生物聚合物，
    所述生物聚合物选自多核苷酸、多肽、多糖和脂质中的一种，优选为多核苷酸，所述多核苷酸包括DNA和/或RNA。
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