WO2023130839A1 - 成膜支架、生物芯片、装置、制备方法及其应用 - Google Patents

成膜支架、生物芯片、装置、制备方法及其应用 Download PDF

Info

Publication number
WO2023130839A1
WO2023130839A1 PCT/CN2022/132207 CN2022132207W WO2023130839A1 WO 2023130839 A1 WO2023130839 A1 WO 2023130839A1 CN 2022132207 W CN2022132207 W CN 2022132207W WO 2023130839 A1 WO2023130839 A1 WO 2023130839A1
Authority
WO
WIPO (PCT)
Prior art keywords
film
forming
liquid storage
insulating layer
chamber
Prior art date
Application number
PCT/CN2022/132207
Other languages
English (en)
French (fr)
Inventor
张喆
夏晓翔
宋璐
Original Assignee
成都齐碳科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 成都齐碳科技有限公司 filed Critical 成都齐碳科技有限公司
Publication of WO2023130839A1 publication Critical patent/WO2023130839A1/zh

Links

Images

Classifications

    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0609—Holders integrated in container to position an object
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes

Definitions

  • the application belongs to the technical field of biological detection, and in particular relates to a film-forming scaffold, a method for preparing a film-forming scaffold, a biochip, a method for preparing a molecular membrane, a device for characterizing biomolecules through nanopores, and applications thereof.
  • nanopore gene sequencing technology it is usually necessary to form a molecular membrane on a biochip to embed a nanopore structure on the molecular membrane.
  • biomolecules such as polynucleotides, polypeptides, polysaccharides, or lipids
  • the degree of current blockage caused by analytes of different structures can be measured by measuring electrical signals such as changes in current to analyze and obtain biological Molecular sequence arrangement information or modification information, etc.
  • the molecular membrane is prepared by a scaffold with a cavity, and the film-forming position of the molecular membrane in the cavity is mainly controlled by the position of the interface between the polar and non-polar medium layers.
  • the medium layer interface is convex in the middle, The concave liquid surface on both sides makes it difficult to accurately control the film-forming position of the molecular film in the cavity, and it is also difficult to guarantee the stability of the prepared molecular film.
  • the embodiments of the present application provide a film-forming scaffold, a method for preparing a film-forming scaffold, a biochip, a method for preparing a molecular membrane, a device for characterizing biomolecules through nanopores, and their application, so as to solve the problem of the molecular membrane manufacturing equipment in the related art.
  • the problem of poor stability of the molecular membrane is a problem of poor stability of the molecular membrane.
  • the first aspect of the embodiments of the present application provides a film-forming support for forming a molecular film.
  • the film-forming support includes an insulating base and an insulating layer.
  • the insulating layer is disposed on the insulating base.
  • the insulating layer includes a first insulating layer and a second insulating layer.
  • the first insulating layer is provided with a liquid storage chamber
  • the second insulating layer is provided with a film forming chamber communicating with the liquid storage chamber
  • the film forming chamber and the The liquid storage cavity opens toward the direction away from the insulating base, and the opening size of the film forming cavity gradually increases along the direction from the insulating base to the second insulating layer.
  • the inner surface of the first insulating layer constitutes the liquid storage surface of the liquid storage chamber
  • the inner surface of the second insulating layer constitutes the film forming surface of the film forming chamber
  • the second insulating layer is away from the top of the insulating base.
  • the surface is smoothly connected with the film-forming surface forming the film-forming chamber, and the film-forming surface is smoothly connected with the liquid storage surface forming the liquid storage chamber.
  • the film-forming surface is an inclined surface or an arc-shaped surface.
  • the second insulating layer further includes a supporting protrusion protruding from the film-forming surface.
  • the supporting protrusions are arranged in sequence along the direction from the liquid storage surface to the top surface.
  • each of the plurality of supporting protrusions includes a supporting edge and a rising edge, the rising edge extends along the direction from the insulating base to the second insulating layer, and the supporting edge extends along the horizontal plane where the insulating base is located. Extending, along the direction from the liquid storage surface to the top surface, the distance between the supporting protrusions arranged in sequence and the insulating matrix gradually increases.
  • each of the plurality of support protrusions includes a falling edge and a rising edge, the falling edge is arranged on the side of the rising edge away from the axis of the film forming chamber, and the edge is from the liquid storage surface to the top. In the direction of the surface, the distance from the apex of the rising edge of each supporting protrusion arranged in sequence to the insulating matrix gradually increases.
  • the supporting protrusions include a plurality of first protrusions connected in sequence to the top surface, and second protrusions connecting the first protrusions and the liquid storage surface; In the direction of the surface, the distance from the apex of each first protrusion arranged in sequence to the insulating base gradually increases.
  • the number of second protrusions is multiple, and the plurality of second protrusions are connected in sequence; along the direction from the axis of the film-forming chamber to the edge of the film-forming chamber, the apex of each second protrusion on the same plane.
  • the film-forming support further includes a plurality of micro-protrusions, and the plurality of micro-protrusions are provided at least on any one of the film-forming surface, the liquid storage surface and the supporting protrusions.
  • the liquid storage surface forming the liquid storage chamber is arranged along a direction from the insulating base to the second insulating layer.
  • the liquid storage chamber includes an upper chamber and a lower chamber, and the film forming chamber, the upper chamber, and the lower chamber are connected in sequence.
  • the opening size gradually decreases.
  • the maximum included angle between the inner wall of the upper chamber and the plane where the insulating base is located is ⁇ , where 1° ⁇ 75°.
  • a plurality of liquid storage chambers are arranged in an array on the first insulating layer, a plurality of film-forming chambers are arranged in an array on the second insulating layer, and adjacent film-forming chambers are arranged on the second insulating layer.
  • the first channel of the layer is connected;
  • the adjacent liquid storage chambers communicate with each other through the second channel provided in the first insulating layer.
  • the embodiment of the present application also provides a method for preparing a film-forming stent, including:
  • An insulating base is provided, and a first insulating layer having a liquid storage cavity is prepared on the insulating base through a photolithography process;
  • the photoresist layer is etched through a mask with a preset pattern to form a hollow area in the photoresist layer connected to the liquid storage chamber, the hollow area is a film-forming cavity corresponding to the preset pattern, and the preparation A film-forming scaffold as described above was obtained.
  • the embodiment of the present application also provides a method for preparing a film-forming stent, including:
  • the stent template was reversed and transferred using imprinting technology to prepare the above-mentioned film-forming stent.
  • the embodiment of the present application also provides a biochip, including:
  • the electrodes are arranged in the liquid storage chamber, and the electrodes can be conductively connected with the liquid in the liquid storage chamber, and can be connected with the external circuit of the biochip.
  • the embodiment of the present application also provides a method for preparing a molecular membrane, including:
  • a molecular film is formed at the film-forming cavity.
  • the embodiment of the present application also provides a biomolecular characterization device, including the above-mentioned film-forming scaffold, the film-forming scaffold prepared by the above-mentioned method, or the above-mentioned biochip.
  • the embodiment of the present application also provides the application of the above-mentioned film-forming scaffold, the above-mentioned biochip, and biomolecular characterization device in biomolecular characterization or preparation of products for biomolecular characterization.
  • the second insulating layer can provide a certain support force to the molecular film in the vertical direction, avoid or reduce the liquid flow in the film-forming cavity, optimize the shape of the liquid surface, and affect the relative position of the molecular film and the film-forming support; It can also increase the contact area between the molecular film and the second insulating layer in the film-forming cavity by preparing a structure with gradually increasing opening size; since the second insulating layer can provide support for the molecular film, it can maintain polar solvents and non-polar solvents.
  • the formed oil-water interface allows the position of the molecular film to be precisely fixed.
  • Fig. 1 is a schematic cross-sectional structure diagram of a film-forming stent provided by an embodiment of the present application
  • Fig. 2 is the enlarged structural diagram of part A shown in Fig. 1;
  • Fig. 3 is a partial cross-sectional structural schematic diagram of a film-forming stent provided by an embodiment of the present application
  • Fig. 4 is a partial cross-sectional structural schematic diagram of a film-forming stent provided by another embodiment of the present application.
  • Fig. 5 is a partially enlarged structural schematic diagram of the film-forming stent shown in Fig. 4;
  • Fig. 6 is a partial cross-sectional structural schematic diagram of a film-forming stent provided by another embodiment of the present application.
  • Fig. 7 is a partially enlarged structural schematic diagram of the film-forming stent shown in Fig. 6;
  • Fig. 8 is a partial cross-sectional structural schematic diagram of a film-forming stent provided in yet another embodiment of the present application.
  • Fig. 9 is a partial cross-sectional structural schematic diagram of a film-forming stent provided by an embodiment of the present application.
  • Fig. 10 is a schematic cross-sectional structure diagram of a film-forming stent provided by another embodiment of the present application.
  • Fig. 11 is a schematic cross-sectional structure diagram of a film-forming stent provided by another embodiment of the present application.
  • Fig. 12 is a schematic cross-sectional structure diagram of a film-forming stent provided by another embodiment of the present application.
  • Fig. 13 is a schematic flow chart of a method for preparing a film-forming stent provided by an embodiment of the present application
  • Fig. 14 is a partial structural schematic diagram during the preparation process of the film-forming stent provided by an embodiment of the present application.
  • Fig. 15 is a partial structural schematic diagram during the preparation process of the film-forming stent provided by another embodiment of the present application.
  • Fig. 16 is a schematic structural diagram during the preparation of the film-forming scaffold provided by another embodiment of the present application.
  • Fig. 17 is a schematic structural diagram of a scanning electron microscope of a film forming cavity provided by an embodiment of the present application.
  • FIG. 18 is a schematic structural diagram of a scanning electron microscope of a film forming chamber provided by another embodiment of the present application.
  • Film-forming support 1. Insulating layer; 11. First insulating layer; 111. Liquid storage surface; 12. Second insulating layer; 121. Top surface; 123. Film-forming surface; 124. Supporting protrusions; 1241. 1242, second protrusion; 1243, support edge; 1244, rising edge; 1245, falling edge; 125, micro protrusion; 13, liquid storage chamber; 131, upper chamber; 132, lower chamber ; 14, film-forming cavity; 2, insulating substrate; 3, electrode;
  • orientation terms in the description of the application are only for the convenience of describing the application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as limiting the scope of the application. limit.
  • the embodiment of the present application provides a film-forming scaffold.
  • the film-forming support 10 includes an insulating base 2 and an insulating layer 1, the insulating layer 1 is arranged on the insulating base 2, the insulating layer 1 includes a first insulating layer 11 and a second insulating layer 12, and the insulating base 2.
  • the first insulating layer 11 and the second insulating layer 12 are stacked in sequence, the first insulating layer 11 is provided with a liquid storage chamber 13; the second insulating layer 12 is provided with a film forming chamber 14 communicating with the liquid storage chamber 13, The opening of the film forming chamber 14 and the liquid storage chamber 13 faces away from the insulating substrate 2 , and the opening size of the film forming chamber 14 gradually increases along the direction from the insulating substrate 2 to the second insulating layer 12 .
  • the film-forming support 10 in the present application is used to prepare a molecular membrane, specifically, it can be a molecular membrane with an amphiphilic molecular layer, and a plurality of nanopore structures connecting both sides of the molecular membrane are arranged in the amphiphilic molecular layer, and the molecular membrane can be Applied in biomolecular characterization based on nanopore sequencing technology.
  • Biomolecules can specifically be one of polynucleotides, polypeptides, polysaccharides and lipids, and polynucleotides include DNA and/or RNA.
  • the film-forming cavity 14 formed on the film-forming support 10 is a cavity with an opening, and reagents for preparing molecular films can be added into the film-forming cavity 14 through the opening, or into the liquid storage cavity 13 through the film-forming cavity 14 .
  • the insulating base 2 , the first insulating layer 11 and the second insulating layer 12 are arranged in sequence along the vertical direction, and the opening is arranged opposite to the insulating base 2 , which facilitates the preparation and use of the film-forming support 10 .
  • the liquid storage chamber 13 can accommodate polar solvents, such as buffer solutions used to prepare molecular membranes, specifically phosphate buffer solutions, HEPES buffer solutions containing KCl or NaCl, CAPS buffer solutions containing KCl or NaCl, and the like. Control the liquid height of the polar solvent in the liquid storage chamber 13, so that the newly added non-polar solvent can be distributed on the surface of the polar solvent and form a liquid film in the film-forming chamber 14, and the non-polar solvent can be used to dissolve amphiphilic
  • the non-polar solvent of the material such as silicone oil, can specifically be methylphenyl silicone oil, polydimethylsiloxane, etc., to drive away the polar solvent, that is, to complete the process of "oil driving water".
  • a polar solvent is added to drive the non-polar solvent of the amphiphilic material, that is, the process of "water driving oil” is completed.
  • a structure of polar solvent-non-polar solvent-polar solvent can be formed in the film-forming chamber 14, and the non-polar solvent of the amphiphilic material is sandwiched between two layers of polar solvent to form a molecular film.
  • a part of the molecular film is suspended in the film-forming chamber 14 , and a part is attached to the wall of the film-forming chamber 14 , that is, to the inner surface of the second insulating layer 12 .
  • the specific shapes of the film forming chamber 14 and the liquid storage chamber 13 are not limited, it is only necessary to ensure that the liquid storage chamber 13 can accommodate polar solvents, and a molecular film can be formed in the film forming chamber 14. There is no limitation on the size and shape of the opening, as long as a molecular film can be formed to satisfy the subsequent characterization of biomolecules.
  • the analyte passes through the transmembrane nanopores and causes a change in current or voltage, which can be changed according to the current or voltage information to obtain characterization information for the analyte.
  • characterization information for the analyte.
  • the size information, sequence information, identity information, modification information, etc. of the analyte can be obtained according to the current change information.
  • the high-quality molecular film prepared by the film-forming scaffold 10 is particularly important for the accuracy and timeliness of the subsequent characterization of biomolecules.
  • the insulating base 2 , the first insulating layer 11 and the second insulating layer 12 are made of the same material for the convenience of preparation. Of course, three different materials can also be used to prepare separately.
  • the first insulating layer 11 and the second insulating layer 12 can be sequentially formed on the insulating substrate 2 by a deposition process, such as chemical vapor deposition or plasma enhanced deposition process. It can be understood that the insulating base 2 , the first insulating layer 11 and the second insulating layer 12 may all be made of dielectric materials, such as silicon dioxide.
  • the nanopore sequencing technology needs to form a potential difference on both sides of the molecular membrane, and the electrode 3 can be further provided to form a potential difference on both sides of the molecular membrane.
  • Forming the film-forming stent 10 makes it possible for the molecular film prepared by the film-forming stent 10 to have the possibility of electrical characterization testing and use in a polar liquid environment.
  • the inner surface of the first insulating layer 11 constitutes the liquid storage surface 111 of the liquid storage chamber 13
  • the inner surface of the second insulating layer 12 constitutes the film-forming surface 123 of the film-forming chamber 14, and the film-forming surface 123 may specifically be a slope,
  • the curved surface, or the surface with structures such as supporting steps, protrusions, etc. compared with the surface arranged vertically to the insulating substrate 2 in the related art, the second insulating layer 12 provided by the application can provide the molecular film in the vertical direction.
  • a certain supporting force can avoid or reduce the flow of liquid in the film-forming cavity 14, optimize the shape of the liquid surface, and affect the relative position of the molecular film and the film-forming support 10; by setting the film-forming cavity 14 as a structure whose opening size gradually increases, Thereby, the contact area between the molecular film and the first insulating layer 11 in the film forming chamber 14 can be increased; since the first insulating layer 11 can provide support for the molecular film, the oil-water interface formed by the polar solvent and the non-polar solvent is maintained, that is, the formation of The position of the molecular membrane can be precisely fixed.
  • the top surface 121 of the second insulating layer 12 away from the insulating base 2 is smoothly connected with the film-forming surface 123 forming the film-forming chamber 14, and the film-forming surface 123 is connected with the liquid storage surface forming the liquid storage chamber 13 111 smooth connection.
  • the top surface 121 can be a plane, or a curved surface with a certain curvature.
  • the film-forming surface 123 is an inclined plane, that is, the plane where the film-forming surface 123 is located intersects the direction perpendicular to the insulating substrate 2 at an angle, and the film-forming chamber 14 formed by the film-forming surface 123 is in the shape of a bell mouth.
  • the film-forming surface 123 is an arc-shaped surface.
  • the arc surface can be a concave surface as shown in FIG. 3 , or a convex surface, and those skilled in the art can select a suitable type and radian of the arc surface according to needs.
  • the film-forming surface 123 is an inclined surface or an arc-shaped surface, it can provide a certain supporting force for the molecular film, and at the same time, the flat inclined surface and the arc-shaped surface can avoid piercing the molecular film and improve the stability of the molecular film.
  • the second insulating layer 12 further includes a supporting protrusion 124 protruding from the film-forming surface 123 .
  • the set support protrusion 124 can hinder the flow of liquid in the cavity, thereby improving the stability of the prepared molecular film; at the same time, the prepared molecular film can be completely attached to the entire support protrusion 124, thereby increasing the molecular film and the second insulating layer 12.
  • the contact area is favorable for the second insulating layer 12 to fix the molecular film.
  • the number of support protrusions 124 may be multiple, and the plurality of support protrusions 124 are sequentially arranged along the direction from the liquid storage surface 111 to the top surface 121 .
  • the specific shape of each supporting protrusion 124 is not limited, and the shapes of the supporting protrusions 124 arranged in sequence may be consistent or not, but the surface of each supporting protrusion 124 is a curved surface.
  • each support protrusion 124 in the plurality of support protrusions 124 includes a support edge 1243 and a rising edge 1244, and the rising edge 1244 extends from the insulating base 2 to the second insulating Extending in the direction of the layer 12, the supporting edge 1243 extends along the horizontal plane of the insulating base 2.
  • the distance from the supporting edge 1243 of each supporting protrusion 124 arranged in sequence to the insulating base 2 gradually increases.
  • each support protrusion 124 in the plurality of support protrusions 124 includes a falling edge 1245 and a rising edge 1244 , and the falling edge 1245 is disposed far from the film forming edge 1244 .
  • the distance from the apex of the ascending edge 1244 of each supporting protrusion 124 arranged in sequence to the insulating base 2 gradually increases.
  • the axis x of the film forming chamber 14 is set along the vertical direction, and the apex of the rising edge 1244 is the end of the rising edge 1244 away from the insulating base 2 .
  • Neither the rising edge 1244 nor the falling edge 1245 is parallel to the vertical direction.
  • Different supporting protrusions 124 are connected in a plane parallel to the insulating base 2 to form an annular protrusion disposed along the film forming chamber 14, and different supporting protrusions 124 are connected in different planes to form multiple annular protrusions.
  • the supporting protrusion 124 includes a plurality of first protrusions 1241 connected in sequence with the top surface 121 , and a second protrusion 1242 connecting the first protrusions 1241 and the liquid storage surface 111 , the first protrusions 1241 is connected to the top surface 121 of the second insulating layer 12; along the direction from the liquid storage surface 111 to the top surface 121, the distance from the apex of each sequentially arranged first protrusion 1241 to the insulating base 2 gradually increases.
  • the vertices of the plurality of first protrusions 1241 are arranged along a certain slope, so that no matter which first protrusion 1241 the liquid level of the polar solvent corresponds to, the corresponding first protrusion 1241 can
  • the molecular film floating on the polar solvent is supported and fixed, and the aforementioned "corresponding" means that the first protrusion 1241 is in contact with the edge of the liquid surface of the polar solution.
  • the number of second protrusions 1242 is multiple, and the plurality of second protrusions 1242 are connected in sequence; along the direction from the axis x of the film forming chamber 14 to the edge of the film forming chamber 14, the apex of each second protrusion 1242 On the same plane, that is, the plurality of second protrusions 1242 form a concavo-convex structure in the same plane. Likewise, the plurality of second protrusions 1242 can limit liquid fluctuations, thereby improving the stability of the molecular film.
  • the film-forming support 10 further includes micro-protrusions 125 , and a plurality of micro-protrusions 125 are provided on at least any one of the film-forming surface 123 , the liquid storage surface 111 and the supporting protrusions 124 .
  • the micro-protrusions 125 can be arranged sequentially at intervals, or can be arranged irregularly.
  • the distance between adjacent micro-protrusions 125 is 0.1-10 ⁇ m, and the size of a single micro-protrusion 125 is 5 nm-50 nm.
  • the micro-protrusion 125 can be in the shape of semicircular dots, cylinders with a certain length, tentacles, and the like.
  • the micro-protrusions 125 may be disposed on the film-forming surface 123 , the supporting protrusions 124 and the liquid storage surface 111 at the same time. By setting the micro-protrusions 125, the fluctuation of the liquid is further restricted, thereby improving the stability of the molecular film.
  • the liquid storage surface 111 forming the liquid storage chamber 13 is arranged along the direction from the insulating base 2 to the second insulating layer 12 , that is, the liquid storage surface 111 can be arranged perpendicular to the insulating base 2 .
  • the shape of the cavity surrounded by the film-forming surface 123 and the liquid storage surface 111 may be consistent or inconsistent.
  • the cross-section of the film-forming chamber 14 surrounded by the film-forming surface 123 has an obtuse angle, an acute angle or a right angle, specifically, any of the obtuse angle, acute angle or right angle can be formed.
  • the molecular film formed by the polar solvent has a tendency to gather toward the inner wall at a certain angle in the film-forming cavity 14, so this region can relatively stably pull the molecular film, and has a relatively stable supporting effect on the molecular film, so The formed molecular film is more stable and has a higher film formation rate.
  • the cross-section of the film-forming chamber 14 may specifically be circular, elliptical, or polygonal such as triangular, rectangular, five-pointed star or polygonal star. It can be understood that the above-mentioned cross-section is a centrosymmetric structure, which can improve the supporting effect of the film-forming surface 123 on the molecular film.
  • the shape of the liquid storage chamber 13 is not limited, and may be polygonal or irregular.
  • the shape of the liquid storage chamber 13 is a polygonal column or an elliptical column, and may be cylindrical.
  • the shape of the liquid storage cavity 13 is cylindrical, it is convenient to process the film-forming stent 10 .
  • the cross-section of the film-forming chamber 14 can be greater than, equal to or smaller than the cross-section of the liquid storage chamber 13, that is, the orthographic projection of the film-forming chamber 14 to the liquid storage chamber 13 covers the liquid storage chamber 13, or the edge and the edge of the liquid storage chamber 13 overlap, or fall into the liquid storage chamber 13.
  • the orthographic projection of the film-forming chamber 14 to the liquid storage chamber 13 coincides with the liquid storage chamber 13, that is, the cross-section of the film-forming chamber 14 is equal to the cross-section of the liquid storage chamber 13, so that the liquid storage chamber 13 and
  • the film-forming chamber 14 is more convenient, improves the yield and consistency of the film-forming stent 10 , and is conducive to improving the film-forming stability and film-forming rate of the molecular film in the film-forming chamber 14 .
  • the liquid storage chamber 13 can be prepared as a structure in which the size is sequentially reduced or gradually expanded along the direction from the film formation chamber 14 to the liquid storage chamber 13 .
  • the liquid storage chamber 13 includes an upper chamber 131 and a lower chamber 132, and the film forming chamber 14, the upper chamber 131 and the lower chamber 132 are connected in sequence, along the direction from the insulating base 2 to the second chamber.
  • the opening size of the upper chamber 131 gradually decreases. Therefore, it has a larger liquid storage capacity, and at the same time, the size of the opening connected to the film-forming chamber 14 is smaller, so that the exposed size of the film-forming liquid surface is smaller and the movement of the liquid surface is reduced.
  • the extending direction of the liquid storage surface 111 can be varied, and have different angles with the plane where the insulating base 2 is located, forming liquid storage chambers 13 with different structures to meet different requirements.
  • the maximum included angle between the inner wall surface of the upper chamber 131 and the horizontal plane is ⁇ , wherein 1° ⁇ 75°, preferably 5° ⁇ 60°, more preferably 15° ° ⁇ 55°.
  • the maximum angle ⁇ between the film-forming surface 123 and the horizontal plane is 10° to 80°, preferably 30° to 60°, or the maximum angle ⁇ between the line connecting the vertices of two adjacent supporting protrusions 124 and the horizontal plane 10° to 80°, preferably 30° to 60°.
  • the inner wall surface of the upper chamber 131 may present a straight line as shown in FIG. A protruding curve in a direction close to the insulating base 2 ; or, as shown in FIG. 12 , a protruding curve in a direction away from the insulating base 2 . That is, the inner wall surface of the upper chamber 131 may specifically be an inclined plane or a curved surface. Please refer to FIG. 11 and FIG. 12 , the dotted line in the figure is a tangent line at a certain point on the inner wall of the upper chamber 131 .
  • each point of the curved surface is used as a tangent line, and the maximum angle between each tangent line and the plane where the insulating matrix 2 is located is ⁇ . That is, the included angle ⁇ in the embodiment of the present application includes the meaning that no matter whether the inner wall surface forming the upper chamber 131 is a plane or a curved surface, the maximum angle formed between the tangent surface of the inner wall surface and the plane where the substrate is located is the included angle ⁇ .
  • Setting the inner wall of the upper chamber 131 as an inclined plane or curved surface can maintain a certain shape of the polar liquid in the liquid storage chamber 13, which is conducive to the stability of the polar liquid in the liquid storage chamber 13 and is conducive to product transportation and long-term storage.
  • one liquid storage chamber 13 may be provided in the first insulating layer 11, and one film forming chamber 14 may be provided in the second insulating layer 12 correspondingly, or multiple liquid storage chambers may be provided in the first insulating layer 11.
  • the cavities 13 correspond to a plurality of film-forming cavities 14 provided in the second insulating layer 12, which are not limited here and are selected according to specific requirements.
  • the plurality of liquid storage chambers 13 can be regularly arranged in an array on the first insulating layer 11, and the plurality of film formation chambers 14 It can be regularly arranged in an array on the second insulating layer 12 to increase the number of film forming chambers 14 and liquid storage chambers 13 distributed in a unit area.
  • irregular arrangements may also be made, and there is no limitation here.
  • one film-forming scaffold 10 can prepare multiple molecular films at the same time, so as to carry out the characterization of multiple biomolecules and improve the efficiency of the detection work.
  • the adjacent film-forming chambers 14 are communicated through the first channel, that is, the ions or molecules in the liquid contained in the film-forming chambers 14 can diffuse through the channels, which is conducive to maintaining the balance of the liquid concentration in each film-forming chamber 14 , it is convenient to add the solution into the film-forming support 10 .
  • the adjacent liquid storage chambers 13 can also be communicated through the second channel, and the ions or molecules in the liquid contained in the liquid storage chambers 13 can diffuse through the second channel, which is conducive to maintaining the balance of the liquid concentration in each liquid storage chamber 13 .
  • At least one of adjacent liquid storage chambers 13 and adjacent film forming chambers 14 are mutually blocked. That is, the liquid storage chambers 13 of the same film-forming support 10 are not connected to each other, and the film-forming chambers 14 of the same film-forming support 10 are not connected to each other, which is conducive to maintaining each liquid storage chamber 13 and the corresponding film-forming chamber 14 The independence of sequencing work will not interfere with each other.
  • the embodiment of the present application provides a method for preparing a film-forming stent, including:
  • S1 also includes: setting blind holes on the insulating substrate through a photolithography process, and then setting electrodes in the blind holes through a metal deposition process, so that the electrodes can be distributed on the insulating substrate.
  • photoresist 20 and/or dry film are coated on insulating substrate 2 and each electrode 3; See Fig. 14(c) and Fig. 14(d), pass
  • the photolithography process uses the liquid storage cavity mask 30 to pattern the photoresist and/or dry film to form the first insulating layer 11 with the liquid storage cavity 13, the position of the liquid storage cavity 13 corresponds to the electrode 3, This allows the electrode 3 to be in contact with the solution contained in the liquid storage chamber 13 .
  • the ratio of the height of the liquid storage chamber 13 to the diameter of the liquid storage chamber 13 is (0.8-3):1.
  • a photoresist material is coated on the end of the first insulating layer 11 away from the insulating base 2 , and solidified photoresist layer 40 is formed after being cured by light.
  • the photoresist layer 40 is prepared from a photoresist material.
  • the photoresist material refers to that after the polymer material is irradiated with light, the molecular structure changes from linear soluble to bulk insoluble, thereby producing resistance to solvents. corrosion ability.
  • the photoresist material may be dichromate photoresist, polyvinyl cinnamate photoresist, or the like.
  • the preset pattern may specifically be a 2D pattern distributed on the mask, or a 3D pattern with a three-dimensional structure.
  • the mask can specifically be a pre-prepared reusable mask plate, or a mask layer formed by coating on the photoresist layer.
  • the preset pattern is a 3D pattern with a three-dimensional structure.
  • S3 can include:
  • the forward structure is a 3D pattern previously designed by those skilled in the art according to the structure of the film-forming cavity.
  • the photoresist 50 in S31 can adopt positive photoresist, at this time through gray scale photolithography process and utilize photolithography mask plate 60 , so that the exposure rates of different positions in the photoresist 50 are different, so as to form a 3D pattern on the photoresist 50 , that is, the forward structure 501 .
  • the photoresist with the forward structure 501 can be used as a mask to perform anisotropic etching and/or isotropic etching on the photoresist layer 40, so that the photoresist
  • the etching layer 40 has a hollowed out area corresponding to the forward structure 501 , and the hollowed out area is the film-forming cavity 14 to be prepared, that is, a film-forming scaffold having the film-forming cavity 14 is prepared.
  • Anisotropic etching is an etching method that exhibits different etching rates on different crystallographic planes, and isotropic etching generally refers to an etching method that exhibits the same etching rate on different crystallographic planes.
  • Those skilled in the art can choose different etching methods according to needs, so as to process the photoresist layer 40 into the second insulating layer 12 with the film forming cavity 14 .
  • the preset pattern may specifically be a 2D pattern distributed on the mask, and S3 includes:
  • the photoresist layer 40 is etched by using the photoresist 50 having a two-dimensional planar pattern 502 as a mask, so that according to the photolithography
  • the planar pattern 502 on the glue 50, the etching selectivity ratio between materials, etc., are processed on the photoresist layer 40 to obtain a hollow area, and the above-mentioned film-forming support is prepared.
  • the mask layer used for processing the photoresist layer 40 may be a two-dimensional planar patterned photoresist and/or a patterned inorganic material film layer.
  • S3 includes:
  • the photoresist layer can specifically be an organic polymer, such as epoxy resin, trimethacrylate, imide, and polycarbonate;
  • the mask can specifically be a mask chrome plate made of quartz. , metal hollow mask, etc.
  • the mask When performing excimer laser etching, the mask can be placed between the excimer laser and the photoresist layer, and the laser radiates ultraviolet photons to partially decompose the photoresist layer to form a film with the required structure stand.
  • the present application also provides another method for preparing a film-forming stent, including:
  • the scaffold template is reversed and transferred by embossing technology, and the above-mentioned film-forming scaffold structure is prepared.
  • the stent template is a reverse structure mold prepared in advance according to the required structure of the film-forming stent, and the stent template can specifically be a metal mold formed by electroforming.
  • Fig. 16(a) when performing over-mold transfer by imprinting technology, an insulating substrate 2 with a circuit structure can be prepared in advance; please refer to Fig. 16(b), Fig. 16(c) and Fig.
  • the thickness of the embossing glue 60 is the same as the sum of the thicknesses of the first insulating layer 11 and the second insulating layer 12 that need to form a film support, press the support template 70 on its surface, use
  • the way of pressurization makes the reverse structure of the scaffold template 70 reversely transferred to the embossing glue 60; please refer to FIG.
  • the photoresist removed by excimer laser etching can be used.
  • first insulating layer and the second insulating layer are obtained, and the insulating substrate, the first insulating layer and the second insulating layer are sequentially bonded to obtain a film-forming support.
  • the method of preparing the membrane-forming stent is not limited to the methods listed above.
  • the above-mentioned membrane-forming scaffold is prepared by the above-mentioned method, so it has the same technical effect, and will not be repeated here.
  • FIG. 17 and FIG. 18 are schematic diagrams of the scanning electron microscope structure of the film-forming cavity prepared by the photolithography process in this application, wherein the film-forming cavity shown in FIG. 17 has an arc-shaped film-forming surface 123, and the film-forming cavity shown in FIG. 18
  • the cavity has a ring-shaped protrusion formed by a second protrusion 1242 and a plurality of first protrusions 1241 connected in sequence.
  • the embodiment of the present application provides a biochip.
  • the biochip includes an electrode 3 and the film-forming support provided by any one of the above-mentioned embodiments.
  • the electrode 3 is arranged at the bottom of the liquid storage chamber 13, and can The liquid in the liquid storage cavity 13 is electrically connected, and can be connected with an external circuit of the biochip.
  • the electrode 3 is used to form a potential difference on both sides of the molecular film, and the film-forming support and the electrode 3 are integrated into a whole, and processed together to form a biochip, which further improves the structural compactness of the biomolecular characterization device.
  • a molecular film is formed at the film-forming cavity.
  • the electrode 3 at the bottom of the liquid storage chamber 13 can only be electrically conductively connected with the conductive liquid in the liquid storage chamber 13, and be electrically conductively connected with the conductive liquid on the upper side of the molecular membrane through an external circuit of the biochip, and the electrodes 3 of the two can be set. On the contrary, a potential difference is generated on both sides of the molecular membrane.
  • the electrode 3 it is also possible to set the electrode 3 to include a part that is conductively connected to the conductive liquid in the liquid storage chamber 13, and to include a part that is conductively connected to the liquid on the top of the film forming chamber 14.
  • the polarities of the electrodes 3 in the two parts are opposite. A potential difference is generated on both sides of the molecular membrane.
  • the electrodes 3 can be disposed on the surface of the insulating base 2 close to the first insulating layer 11 , or can be embedded in the structure of the insulating base 11 , which is not limited here.
  • a method for preparing a molecular membrane comprising:
  • a molecular film is formed at the film-forming cavity.
  • the preparation method may specifically include:
  • the non-polar reagent pre-coated at this time may be a non-polar reagent containing amphiphilic molecules for forming a molecular film, or other non-polar reagents.
  • Pre-coating the non-polar reagent is beneficial to improve the adhesion and smoothness of the movement of the bubbles on the surface of the biochip, and improve the film-forming efficiency of the molecular film.
  • the polar reagent may be a non-polar reagent containing amphiphilic molecules used to form a molecular film; a second polar solvent is added into the film-forming cavity, so that the non-polar solvent forms a molecular film in the film-forming cavity.
  • the first polar solvent and the second polar solvent may be the same or different.
  • the film-forming scaffold, biochip and nanopore device for characterizing biomolecules provided in the embodiments of the present application are not limited to the film-forming method provided in the fourth aspect during use, and can also be prepared by other methods.
  • Membrane scaffolds and molecular membranes are not limited to the film-forming method provided in the fourth aspect during use, and can also be prepared by other methods.
  • the embodiment of the present application provides a biomolecular characterization device, including the film-forming scaffold provided in any one of the above embodiments, and the latter includes the biochip provided in any one of the above embodiments.
  • the biomolecular characterization device provided by the embodiments of the present application has the same technical effect because it uses the film-forming scaffold provided by any one of the above embodiments, or adopts the biochip provided by any one of the above embodiments, so it will not be repeated here.
  • the embodiment of the present application provides a film-forming scaffold provided by any one of the above embodiments, the film-forming scaffold prepared by any one of the above embodiments, the biochip provided by any one of the above embodiments, and any one of the above embodiments provides
  • biomolecules include biopolymers, and biopolymers are selected from polynucleotides, polypeptides, polysaccharides and lipids, which can be Selected as polynucleotides, polynucleotides include DNA and/or RNA.
  • biomolecular characterization since the film-forming scaffold, biochip, and biomolecular characterization device provided by any one of the above embodiments are used, they have the same technical effect and will not be repeated here.
  • B corresponding to A means that B is associated with A, and B can be determined according to A.
  • determining B according to A does not mean determining B only according to A, and B may also be determined according to A and/or other information.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Clinical Laboratory Science (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Dispersion Chemistry (AREA)
  • Sustainable Development (AREA)
  • Nanotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Medical Informatics (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

一种成膜支架（10）、生物芯片、装置、制备方法及其应用，成膜支架（10）包括绝缘基体（2）和绝缘层（1），绝缘层（1）设置于绝缘基体（2）上，绝缘层（1）包括第一绝缘层（11）和第二绝缘层（12），绝缘基体（2）、第一绝缘层（11）和第二绝缘层（12）依次叠设，第一绝缘层（11）中设置有储液腔（13），第二绝缘层（12）中设置有与储液腔（13）连通的成膜腔（14），成膜腔（14）和储液腔（13）朝向背离绝缘基体（2）的方向开口，沿从绝缘基体（2）至第二绝缘层（12）的方向，成膜腔（14）的开口尺寸逐渐增大。成膜支架（10）制备的分子膜稳定性好。

Description

成膜支架、生物芯片、装置、制备方法及其应用
相关申请的交叉引用
本申请要求享有于2022年1月10日提交的名称为“成膜支架、生物芯片、装置、制备方法及其应用”的中国专利申请202210022647.9的优先权，该申请的全部内容通过引用并入本文中。
技术领域
本申请属于生物检测技术领域，尤其涉及一种成膜支架、成膜支架的制备方法、生物芯片、分子膜的制备方法、纳米孔表征生物分子的装置及其应用。
背景技术
在纳米孔基因测序技术领域，通常需要在生物芯片上形成分子膜，以在分子膜上嵌入纳米孔结构。当生物分子(例如多核苷酸、多肽、多糖或脂质)通过纳米孔时，由于不同结构的分析物所引起的电流阻断程度不同，从而可以通过测量电信号如电流的变化，分析获取生物分子的序列排布信息或修饰信息等。
相关技术中，分子膜通过具有腔体的支架制备而成，分子膜在腔体中的成膜位置主要依靠极性与非极性介质层界面所在位置控制，但由于介质层界面为中间凸、两侧凹的液面，导致分子膜在腔体中的成膜位置难以精确控制，制备得到的分子膜的稳定性也难以保障。
发明内容
本申请实施例提供了一种成膜支架、成膜支架的制备方法、生物芯片、分子膜的制备方法、纳米孔表征生物分子的装置及其应用，以解决相关技术中分子膜制造设备制备得到的分子膜稳定性不佳的问题。
本申请实施例的第一方面提供一种成膜支架，用于形成分子膜，成膜支架包括绝缘基体和绝缘层，绝缘层设置于绝缘基体上，绝缘层包括第一绝缘层和第二绝缘层，绝缘基体、第一绝缘层和第二绝缘层依次叠设，第一绝缘层中设置有储液腔，第二绝缘层中设置有与储液腔连通的成膜腔，成膜腔和储液腔朝向背离绝缘基体的方向开口，沿从绝缘基体至第二绝缘层的方向，成膜腔的开口尺寸逐渐增大。
根据本申请一个方面的实施例，第一绝缘层的内表面构成储液腔的储液表面，第二绝缘层的内表面构成成膜腔的成膜表面，第二绝缘层背离绝缘基体的顶面与形成成膜腔的成膜表面平滑连接，成膜表面与形成储液腔的储液表面平滑连接。
根据本申请一个方面的实施例，成膜表面为倾斜面或弧形面。
根据本申请一个方面的实施例，第二绝缘层还包括自成膜表面凸出设置的支撑凸起。
根据本申请一个方面的实施例，支撑凸起的数量为多个，多个支撑凸起沿从储液表面到顶面的方向依次设置。
根据本申请一个方面的实施例，多个支撑凸起中的每个支撑凸起包括支撑沿和上升沿，上升沿沿从绝缘基体至第二绝缘层的方向延伸，支撑沿沿绝缘基体所在水平面延伸，沿从储液表面到顶面的方向，依次设置的各个支撑凸起的支撑沿到绝缘基体的距离逐渐增加。
根据本申请一个方面的实施例，多个支撑凸起中的每个支撑凸起包括下降沿和上升沿，下降沿设置于上升沿远离成膜腔的轴线的一侧，沿从储液表面到顶面的方向，依次设置的各个支撑凸起的上升沿的顶点到绝缘基体的距离逐渐增加。
根据本申请一个方面的实施例，支撑凸起包括与顶面相连的多个依次连接的第一凸起，以及连接第一凸起和储液表面的第二凸起；沿从储液表面到顶面的方向，依次设置的各个第一凸起的顶点到绝缘基体的距离逐渐增加。
根据本申请一个方面的实施例，第二凸起的数量为多个，多个第二凸起依次连接；沿从成膜腔的轴线至成膜腔的边沿方向，各个第二凸起的顶 点在同一平面上。
根据本申请一个方面的实施例，成膜支架还包括多个微凸起，多个微凸起至少设于成膜表面、储液表面和支撑凸起中的任一个。
根据本申请一个方面的实施例，形成储液腔的储液表面沿从绝缘基体到第二绝缘层的方向设置。
根据本申请一个方面的实施例，储液腔包括上腔室和下腔室，成膜腔、上腔室和下腔室依次连通，沿从绝缘基体到第二绝缘层的方向，上腔室的开口尺寸逐渐减小。
可选地，上腔室的内壁面与绝缘基体所在平面之间的最大夹角为α，其中，1°＜α＜75°。
根据本申请一个方面的实施例，多个储液腔呈阵列设置在第一绝缘层，多个成膜腔呈阵列设置在第二绝缘层，相邻成膜腔之间通过设置于第二绝缘层的第一通道连通；
根据本申请一个方面的实施例，相邻储液腔之间通过设置于第一绝缘层的第二通道连通。
第二方面，本申请实施例还提供一种成膜支架的制备方法，包括：
提供绝缘基体，通过光刻工艺在绝缘基体上制备具有储液腔的第一绝缘层；
在第一绝缘层远离绝缘基体的一侧制备固化的光致抗蚀层；
通过具有预设图案的掩膜，对光致抗蚀层进行刻蚀，以在光致抗蚀层形成与储液腔连通的镂空区域，镂空区域为与预设图案对应的成膜腔，制备得到如上述成膜支架。
第三方面，本申请实施例还提供一种成膜支架的制备方法，包括：
制备如上述成膜支架对应反向结构的支架模板；
对支架模板采用压印技术进行翻模转印，制备得到如上述成膜支架。
第四方面，本申请实施例还提供一种生物芯片，包括：
如上述的成膜支架或如上述方法制备得到的成膜支架；
电极，设置于储液腔，电极能够与储液腔内的液体导电连接，且能够与生物芯片外部的电路连接。
第五方面，本申请实施例还提供一种分子膜的制备方法，包括：
使用如上述成膜支架、或如上述方法制备得到的成膜支架、或如上述述的生物芯片，在成膜腔处形成分子膜。
第六方面，本申请实施例还提供一种生物分子表征装置，包括如上述的成膜支架、如上述方法制备得到的成膜支架、或者如上述的生物芯片。
第七方面，本申请实施例还提供上述成膜支架、上述生物芯片、生物分子表征装置在生物分子表征或制备用于生物分子表征的产品中的应用。
本申请提供的成膜支架、成膜支架的制备方法、生物芯片、分子膜的制备方法、纳米孔表征生物分子的装置及其应用中，通过设置开口尺寸逐渐增大的成膜腔，使得第二绝缘层可以在竖直方向上对分子膜提供一定的支撑力，避免或减小成膜腔内液体流动，优化液体表面形状，影响分子膜与成膜支架的相对位置；通过将成膜腔制备成开口尺寸逐渐增大的结构，还可以增加成膜腔内分子膜与第二绝缘层的接触面积；由于第二绝缘层能对分子膜提供支撑力，维持极性溶剂和非极性溶剂形成的油水界面，使得形成分子膜的位置可以精确固定。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对本申请实施例中所需要使用的附图作简单地介绍，显而易见地，下面所描述的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本申请一实施例提供的成膜支架的剖面结构示意图；
图2为图1所示A部分的放大结构图；
图3为本申请一实施例提供的成膜支架的部分剖面结构示意图；
图4为本申请另一实施例提供的成膜支架的部分剖面结构示意图；
图5为图4所示成膜支架的部分放大结构示意图；
图6本申请又一实施例提供的成膜支架的部分剖面结构示意图；
图7为图6所示成膜支架的部分放大结构示意图；
图8为本申请再一实施例提供的成膜支架的部分剖面结构示意图；
图9为本申请一实施例提供的成膜支架的部分剖面结构示意图；
图10为本申请另一实施例提供的成膜支架的剖面结构示意图；
图11为本申请又一实施例提供的成膜支架的剖面结构示意图；
图12为本申请再一实施例提供的成膜支架的剖面结构示意图；
图13为本申请一实施例提供的成膜支架制备方法的流程示意图；
图14为本申请一实施例提供的成膜支架制备过程中的部分结构示意图；
图15为本申请另一实施例提供的成膜支架制备过程中的部分结构示意图；
图16为本申请再一实施例提供的成膜支架制备过程中的结构示意图；
图17为本申请一实施例提供的成膜腔的扫描电镜结构示意图；
图18为本申请另一实施例提供的成膜腔的扫描电镜结构示意图。
附图标记说明：
10、成膜支架；1、绝缘层；11、第一绝缘层；111、储液表面；12、第二绝缘层；121、顶面；123、成膜表面；124、支撑凸起；1241、第一凸起；1242、第二凸起；1243、支撑沿；1244、上升沿；1245、下降沿；125、微凸起；13、储液腔；131、上腔室；132、下腔室；14、成膜腔；2、绝缘基体；3、电极；
20、光刻胶；30、储液腔掩膜板；40、光致抗蚀层；50、光刻胶；501、正向结构；502、平面图案；60、光刻掩膜板。
具体实施方式
下面将详细描述本申请的各个方面的特征和示例性实施例。在下面的详细描述中，提出了许多具体细节，以便提供对本申请的全面理解。但是，对于本领域技术人员来说很明显的是，本申请可以在不需要这些具体细节中的一些细节的情况下实施。下面对实施例的描述仅仅是为了通过示出本申请的示例来提供对本申请的更好的理解。
本申请描述中的方位术语仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合附图对实施例进行详细描述。
第一方面，本申请实施例提供一种成膜支架。如图1和图2所示，成膜支架10包括绝缘基体2和绝缘层1，绝缘层1设置于绝缘基体2上，绝缘层1包括第一绝缘层11和第二绝缘层12，绝缘基体2、第一绝缘层11和第二绝缘层12依次叠设，第一绝缘层11中设置有储液腔13；第二绝缘层12中设置有与储液腔13连通的成膜腔14，成膜腔14和储液腔13朝向背离绝缘基体2的方向开口，沿从绝缘基体2至第二绝缘层12的方向，成膜腔14的开口尺寸逐渐增大。
本申请中的成膜支架10用于制备分子膜，具体可以是具有两亲分子层的分子膜，在两亲分子层中设置有多个连通分子膜两侧的纳米孔结构，该分子膜可应用于基于纳米孔测序技术的生物分子表征中。生物分子具体可以是多核苷酸、多肽、多糖和脂质中的一种，多核苷酸包括DNA和/或RNA。
成膜支架10上形成的成膜腔14为具有开口的腔体，制备分子膜的试剂可以通过该开口加入成膜腔14中，或经过成膜腔14加入储液腔13中。可以理解的是，绝缘基体2、第一绝缘层11和第二绝缘层12沿竖直方向依次设置，开口与绝缘基体2相对设置，方便成膜支架10的制备和使用。储液腔13可以收容极性溶剂，例如用于制备分子膜的缓冲溶液，具体可为磷酸盐缓冲溶液、含有KCl或NaCl的HEPES缓冲溶液、含有KCl或NaCl的CAPS缓冲溶液等。控制储液腔13中极性溶剂的液面高度，使得新加入的非极性溶剂可以分布在极性溶剂表面并在成膜腔14内形成液膜，非极性溶剂可以为溶解两亲性材料的非极性溶剂，如硅油，具体可为甲基苯基硅油、聚二甲基硅氧烷等，对极性溶剂进行驱赶，即完成“油赶水”过程。在此基础上，再通入极性溶剂对两亲性材料的非极性溶剂进行驱赶，即完成“水赶油”过程。此时，成膜腔14内可形成极性溶剂-非极性溶剂-极性溶剂的结构，两亲性材料的非极性溶剂夹在两层极性溶剂之间形成分子膜。该分子膜一部分悬置于成膜腔14内，一部分依附在成膜腔14的壁面上，即依附在第二绝缘层12的内表面上。成膜腔14和储液腔13具体形 状不做限定，仅需保证储液腔13可收容极性溶剂，成膜腔14内可以形成分子膜即可。开口的大小和形状也不做限制，只要能够形成分子膜，满足后续表征生物分子即可。
在成膜支架10上制备具有跨膜纳米孔的分子膜后，可以施加一定的电势，在电势的作用下待分析物从跨膜纳米孔通过并引起电流或电压变化，可根据电流或电压变化信息得到待分析物的表征信息。例如根据电流变化信息得到待分析物的尺寸信息、序列信息、同一性信息、修饰信息等。通过成膜支架10制备得到高质量的分子膜，对后续表征生物分子的准确性和时效性尤为重要。
本申请中绝缘基体2、第一绝缘层11、第二绝缘层12中至少两者的材料相同，以方便制备。当然还可以采用三种不同材料分别制备。可以采用沉积的工艺在绝缘基体2上依次形成第一绝缘层11和第二绝缘层12，如化学气相沉积或者等离子体增强等沉积工艺。可以理解的是，绝缘基体2、第一绝缘层11、第二绝缘层12可以均由介电材料制成，例如二氧化硅。可以理解的是，纳米孔测序技术需要在分子膜的两侧形成电势差，可以进一步设置电极3，以使分子膜两侧形成电势差，电极3具体可以通过结合金属沉积和/或电镀等工艺局部区域化在成膜支架10，使得通过成膜支架10制备的分子膜在极性液体的环境下具备电学表征测试和使用的可能。
本申请中第一绝缘层11的内表面构成储液腔13的储液表面111，第二绝缘层12的内表面构成成膜腔14的成膜表面123，成膜表面123具体可以是斜面、弧面、或是具有支撑台阶、凸起等结构的表面，与相关技术中垂直于绝缘基体2设置的表面相比较，本申请提供的第二绝缘层12可以在竖直方向上对分子膜提供一定的支撑力，避免或减小成膜腔14内液体流动，优化液体表面形状，影响分子膜与成膜支架10的相对位置；通过将成膜腔14设置为开口尺寸逐渐增大的结构，从而可以增加成膜腔14内分子膜与第一绝缘层11的接触面积；由于第一绝缘层11能对分子膜提供支撑力，维持极性溶剂和非极性溶剂形成的油水界面，即形成分子膜的位置可以精确固定。
如图1和图2所示，第二绝缘层12背离绝缘基体2的顶面121与形成 成膜腔14的成膜表面123平滑连接，成膜表面123与形成储液腔13的储液表面111平滑连接。顶面121可以是平面，也可以是具有一定弧度的曲面。通过将各个表面之间设置为平滑连接，避免形成锋利的连接角刺破制备的分子膜，有利于提高制备分子膜的稳定性。
在一实施例中，成膜表面123为倾斜面，即该成膜表面123所在平面与垂直于绝缘基体2的方向呈角度相交设置，成膜表面123形成的成膜腔14呈喇叭口状。如图3所示，在另一实施例中，成膜表面123为弧形面。弧形面可以是如图3所示的凹面，也可以是凸面，本领域技术人员可以根据需要选择合适的弧形面类型和弧度。无论成膜表面123是倾斜面还是弧形面，均能对分子膜提供一定的支撑力，同时平整的倾斜面和弧形面能避免刺破分子膜，提高分子膜的稳定性。
如图4所示，在另一实施例中，第二绝缘层12还包括自成膜表面123凸出设置的支撑凸起124。设置的支撑凸起124可阻碍腔体内液体的流动，从而提高制备的分子膜的稳定性；同时制备的分子膜可以完全附着在整个支撑凸起124上，从而增加分子膜和第二绝缘层12的接触面积，有利于第二绝缘层12固定分子膜。
支撑凸起124的数量可以为多个，多个支撑凸起124沿从储液表面111到顶面121的方向依次设置。每个支撑凸起124的具体形状不做限定，依次设置的多个支撑凸起124的形状可以一致，也可以不一致，但是每个支撑凸起124的表面均为曲面。
如图4和图5所示，在一实施例中，多个支撑凸起124中的每个支撑凸起124包括支撑沿1243和上升沿1244，上升沿1244沿从绝缘基体2至第二绝缘层12的方向延伸，支撑沿1243沿绝缘基体2所在水平面延伸，沿从储液表面111到顶面121的方向，依次设置的各个支撑凸起124的支撑沿1243到绝缘基体2的距离逐渐增加。
如图6和图7所示，在另一实施例中，多个支撑凸起124中的每个支撑凸起124包括下降沿1245和上升沿1244，下降沿1245设置于上升沿1244远离成膜腔14的轴线x的一侧，沿从储液表面111到顶面121的方向，依次设置的各个支撑凸起124的上升沿1244的顶点到绝缘基体2的距 离逐渐增加。成膜腔14的轴线x沿竖直方向设置，上升沿1244的顶点为上升沿1244远离绝缘基体2的末端。上升沿1244和下降沿1245均不与竖直方向平行。不同支撑凸起124在平行于绝缘基体2所在平面内连接，可以形成沿成膜腔14设置的环状凸起，不同支撑凸起124在不同平面内连接，可以形成多个环状凸起。
请参阅图8，支撑凸起124包括与顶面121相连的多个依次连接的第一凸起1241，以及连接第一凸起1241和储液表面111的第二凸起1242，第一凸起1241与第二绝缘层12的顶面121相连；沿从储液表面111到顶面121的方向，依次设置的各个第一凸起1241的顶点到绝缘基体2的距离逐渐增加。在本实施例中，多个第一凸起1241的顶点沿一定的坡度设置，使得无论极性溶剂的液面对应于哪一个第一凸起1241，该对应的第一凸起1241均能对极性溶剂上漂浮的分子膜进行支撑和固定，前述“对应”指第一凸起1241与极性溶液的液面边缘接触。
进一步地，第二凸起1242的数量为多个，多个第二凸起1242依次连接；沿从成膜腔14的轴线x至成膜腔14的边沿方向，各个第二凸起1242的顶点在同一平面上，即多个第二凸起1242在同一平面内形成凹凸结构。同样地，多个第二凸起1242可以限制液体波动，从而提高分子膜的稳定性。
请参阅图9，成膜支架10还包括微凸起125，多个微凸起125至少设于成膜表面123、储液表面111和支撑凸起124中的任一个。该微凸起125可以依次间隔设置，也可以是不规则设置。可选的，相邻微凸起125之间间隔0.1～10μm，单个微凸起125的尺寸为5nm～50nm。微凸起125具体可以是呈半圆凸起的点状、具有一定长度的圆柱体状、触手状等等。微凸起125可以同时设置在成膜表面123、支撑凸起124和储液表面111上。通过设置微凸起125，进一步限制液体波动，从而提高分子膜的稳定性。
进一步地，形成储液腔13的储液表面111沿绝缘基体2到第二绝缘层12方向设置，即储液表面111可以垂直于绝缘基体2设置。当在垂直于绝缘基体2的方向上观察时，成膜表面123和储液表面111围成的空腔形状 可以一致，也可以不一致。在一些实施例中，沿垂直于绝缘基体2的方向，成膜表面123围成的成膜腔14的横截面具有钝角、锐角或直角，具体地，可以形成有钝角、锐角或直角中的任意一种或多种。可以理解的是，极性溶剂形成的分子膜在成膜腔14内具有向一定角度的内壁聚集的趋势，因此该区域能够相对稳定地牵引分子膜，对分子膜具有较为稳定的支撑作用，如此形成的分子膜更加稳定且具有更高的成膜率。该成膜腔14的横截面具体可以是呈圆形、椭圆形，还可以呈三角形、矩形、五角星形或者多角星形等多边形。可以理解的是，上述截面为中心对称结构，可提高成膜表面123对分子膜的支撑效果。
可以理解的是，储液腔13的形状不做限制，可以是多棱柱形，也可以是不规则形状。示例性地，储液腔13的形状为多棱柱形或者椭圆柱形，可选为圆柱形。当储液腔13的形状为圆柱形时，便于成膜支架10的加工。
成膜腔14的横截面可以大于、等于或者小于储液腔13的横截面，即成膜腔14向储液腔13方向的正投影覆盖储液腔13，或者边缘与储液腔13的边缘重叠，或者落入储液腔13内。在一些实施例中，成膜腔14向储液腔13方向的正投影与储液腔13重合，即成膜腔14的横截面等于储液腔13的横截面，如此制备储液腔13和成膜腔14更方便，提高成膜支架10的良品率和一致性，有利于提高成膜腔14内分子膜的成膜稳定性和成膜率。可以根据用户所需储液腔13存储容量、成膜尺寸等因素，将储液腔13制备为沿成膜腔14到储液腔13方向，尺寸依次减缩或依次渐扩结构。
请参阅图10，在一实施例中，储液腔13包括上腔室131和下腔室132，成膜腔14、上腔室131和下腔室132依次连通，沿从绝缘基体2到第二绝缘层12的方向，上腔室131的开口尺寸逐渐减小。从而具有更大的储液容量，同时与成膜腔14连接处的开口尺寸较小，使得成膜液面外露的尺寸较小，减小液面移动。当然储液表面111的延伸方向可以多变，与绝缘基体2所在平面具有不同大小夹角，形成不同结构的储液腔13，以满足不同需求。以绝缘基体2所在平面为水平面，上腔室131的内壁面与水平面之间的最大夹角为α，其中1°＜α＜75°，优选为5°＜α＜60°，更优选为15°＜α＜55°。成膜表面123与水平面之间的最大夹角 β为10°～80°，优选为30°～60°，或两相邻支撑凸起124的顶点的连线与水平面之间的最大夹角β为10°～80°，优选为30°～60°。
沿从绝缘基体2到第二绝缘层12的方向，储液腔13的剖面结构中，上腔室131的内壁面可以如图10所示，呈现直线状态；也可以是如图11，呈现向靠近绝缘基体2方向凸出的曲线状态；或是如图12，呈现向远离绝缘基体2方向凸出的曲线状态。即上腔室131的内壁面具体可以是倾斜的平面，也可以是曲面。请参阅图11和图12，图中虚线为上腔室131的内壁面中的某一点在内壁面的切线。内壁面为曲面时，以曲面的各点做切线，各切线与衬绝缘基体2所在的平面之间的夹角最大值为α。即本申请实施例中的夹角α所包括的含义为无论形成上腔室131的内壁面为平面或曲面，内壁面的切面与衬底所在平面形成的最大夹角为夹角α。将上腔室131的内壁面设置为倾斜的平面或曲面，可维持储液腔13内极性液体具有一定的形貌，有利于储液腔13内极性液体的稳定性，有利于产品运输和长期保存。
可以理解的是，可以在第一绝缘层11中设置一个储液腔13，对应地在第二绝缘层12中设置一个成膜腔14，也可以在第一绝缘层11中设置多个储液腔13，对应的在第二绝缘层12中设置多个成膜腔14，这里不做限制，根据具体需求进行选取。当在一个成膜支架10上设置多个储液腔13和多个成膜腔14时，多个储液腔13可以有规律的呈阵列设置在第一绝缘层11，多个成膜腔14可以有规律的呈阵列设置在第二绝缘层12，以增加单位面积内分布的成膜腔14和储液腔13数量。当然也可以没有规律的排布，这里不做限制。如此一来，一个成膜支架10可以同时制备多个分子膜，以进行多个生物分子的表征工作，提高检测工作的效率。
可选地，相邻成膜腔14之间通过第一通道连通，即成膜腔14内收容的液体中的离子或分子可以通过通道扩散，有利于保持各个成膜腔14内液体浓度的平衡，方便向成膜支架10内添加溶液。同样相邻储液腔13之间也可以通过第二通道连通，储液腔13内收容的液体中的离子或分子可以通过第二通道扩散，有利于保持各个储液腔13内液体浓度的平衡。
在一实施例中，相邻储液腔13、相邻成膜腔14中至少一者相互阻隔。 即同一成膜支架10的各储液腔13之间互不连通，同一成膜支架10的各成膜腔14之间互不连通，有利于保持各储液腔13和对应的成膜腔14测序工作的独立性，互相之间不会产生干扰。
请参阅图13，第二方面，本申请实施例提供一种成膜支架的制备方法，包括：
S1，提供绝缘基体，通过光刻工艺在绝缘基体上制备具有储液腔的第一绝缘层；
S2，在第一绝缘层远离绝缘基体的一侧制备固化的光致抗蚀层；
S3，通过具有预设图案的掩膜，对光致抗蚀层进行刻蚀，例如进行干法刻蚀或激光刻蚀，以在光致抗蚀层形成与储液腔连通的镂空区域，制备得到如上述成膜支架，镂空区域为与预设图案对应的成膜腔。
具体地，在S1之前还包括：通过光刻工艺在绝缘基体上设置盲孔，再通过金属沉积工艺在盲孔中设置电极，使得电极可以分布在绝缘基体上。请参阅图14(a)和图14(b)，在绝缘基体2以及各电极3上方涂覆光刻胶20和/或干膜；请参阅图14(c)和图14(d)，通过光刻工艺利用储液腔掩膜板30对光刻胶和/或干膜进行结构图形化，以形成具有储液腔13的第一绝缘层11，储液腔13的位置与电极3对应，使得电极3可以与储液腔13内收容的溶液接触。储液腔13的高度与储液腔13的直径比为(0.8～3)：1。
请参阅图15(a)，在第一绝缘层11远离绝缘基体2的一端涂覆光致抗蚀材料，光照后固化形成固态的光致抗蚀层40。光致抗蚀层40由光致抗蚀材料制备而成，光致抗蚀材料是指高分子材料经光照辐射后，分子结构从线型可溶性转变为体型不可溶性，从而产生了对溶剂的抗蚀能力。光致抗蚀材料可以是重铬酸盐光敏抗蚀剂、聚乙烯醇肉桂酸酯光敏抗蚀剂等。
S3中，预设图案具体可以是分布在掩膜上的2D图案，也可以是具有立体结构的3D图案。同样掩膜具体可以是预先制备的可重复使用的掩膜板，也可以是光致抗蚀层上涂覆形成的掩膜层。
在一实施例中，预设图案是具有立体结构的3D图案。S3可以包括：
S31，在光致抗蚀层上涂覆一层光刻胶，并利用灰度光刻工艺将光刻 胶制备为具有正向结构的掩膜层；
S32，将具有正向结构的掩膜层作为掩膜，对光致抗蚀层进行刻蚀，以使光致抗蚀层形成与正向结构对应的镂空区域，制备得到如上述成膜支架。
正向结构为本领域技术人员预先根据成膜腔结构设计的3D图案。请参阅图15(b)、图15(c)和图15(d1)，S31中的光刻胶50可以采用正性光刻胶，此时通过灰度光刻工艺并利用光刻掩膜板60，使得光刻胶50中不同位置曝光率不同，从而在光刻胶50上形成3D图案，即正向结构501。
请参阅图15(e)，可以将具有正向结构501的光刻胶作为掩膜，对光致抗蚀层40进行各向异性刻蚀和/或各向同性刻蚀，以使光致抗蚀层40具有该正向结构501对应的镂空区域，该镂空区域为所需制备的成膜腔14，即制备得到具有成膜腔14的成膜支架。各向异性刻蚀为在不同的结晶学平面呈现出不同的腐蚀速率的刻蚀方法，各向同性刻蚀通常是指不同的结晶学平面呈现出相同腐蚀速率的腐蚀方法。本领域技术人员可以根据需要自行选择不同的刻蚀方法，以加工光致抗蚀层40为具有成膜腔14的第二绝缘层12。
在另一实施例中，预设图案具体可以是分布在掩膜上的2D图案，S3包括：
S33，在光致抗蚀层上涂覆一层光刻胶，通过干法反应刻蚀将光刻胶制备为具有平面图案的掩膜层；
S34，将具有平面图案的掩膜层作为掩膜，对光致抗蚀层进行刻蚀，以使光致抗蚀层形成与平面图案对应的镂空区域，制备得到上述成膜支架。
请参阅图15(d2)和图15(e)，上述实施例中通过将具有二维平面图案502的光刻胶50作为掩膜，对光致抗蚀层40进行刻蚀，以根据光刻胶50上的平面图案502、材料间的刻蚀选择比等，在光致抗蚀层40上加工得到镂空区域，制备得到如上述成膜支架。
与S32不同的是，S34中，加工光致抗蚀层40所使用的掩膜层可以是二维平面图形化的光刻胶和/或图形化的无机材料膜层。
在又一实施例中，S3包括：
S35，制备具有预设图案的掩膜，通过激光刻蚀实现预设图案转移到光致抗蚀层，制备得到上述成膜支架。激光刻蚀种类中优选为准分子激光刻蚀。
具体地，光致抗蚀层具体可以是有机高分子聚合物，例如：环氧树脂、三甲基丙烯酸酯、酰亚胺及聚碳酸酯等；掩膜具体可以是石英材质的掩膜铬版、金属镂空掩膜等。进行准分子激光刻蚀时，可以将掩膜放置于准分子激光器和光致抗蚀层之间，通过激光器辐射出紫外光子，将光致抗蚀层进行部分分解，形成所需的结构的成膜支架。
请参阅图16，本申请还提供了另一种成膜支架的制备方法，包括：
S4，制备如上述成膜支架对应反向结构的支架模板；
S5，对支架模板采用压印技术进行翻模转印，制备得到如上述成膜支架结构。
支架模板为预先根据成膜支架所需结构制备的反向结构模具，支架模板具体可以是通过电铸形成的金属模具。请参阅图16(a)通过压印技术进行翻模转印时，可以预先制备具有电路结构的绝缘基体2；请参阅图16(b)、图16(c)图16(d)，在绝缘基体2上涂上压印胶60，该压印胶60的厚度与需要成膜支架的第一绝缘层11和第二绝缘层12的厚度之和相同，将支架模板70压在其表面，采用加压的方式使支架模板70的反向结构反向转移到压印胶60上；请参阅图16(e)，制备得到具有正向结构的成膜支架，即具有第一绝缘层11和第二绝缘层12的成膜支架。S5之后还可以包括：通过干法刻蚀的方法去除残留的压印胶60，以提高底部电极的导通性能和加工精度。当然可以采用准分子激光刻蚀去除的光刻胶。可以理解的是，在一些实施例中，由于成膜腔或储液腔结构复杂，导致翻模转印过程中脱模困难，所以，可以根据所需制备的成膜支架的结构，制备多个不同结构的子模板，再通过各子模板进行翻模转印得到多个零部件，将各零部件键合得到成膜支架。例如，预先制备与第一绝缘层对应反向结构的第一子模板，与第二绝缘层对应反结构的第二子模板，再通过第一子模板和第二子模板进行翻模转印，得到第一绝缘层和第二绝缘层，将绝缘基 板、第一绝缘层和第二绝缘层依次键合得到成膜支架。
制备成膜支架的方式不局限于上述列举的方式，当然采用上述方法制备得到如上成膜支架，因而具有相同的技术效果，这里不再赘述。
请参阅图17和图18，为本申请通过光刻工艺制备得到成膜腔的扫描电镜结构示意图，其中，图17展示的成膜腔具有弧形的成膜表面123，图18展示的成膜腔具有一个第二凸起1242形成的环状凸起和多个依次连接的第一凸起1241。
第三方面，本申请实施例提供一种生物芯片，如图1所示，生物芯片包括电极3和上述任意一实施例提供的成膜支架，电极3设置在储液腔13的底部，能够与储液腔13内的液体导电连接，且能够与生物芯片外部的电路连接。电极3用于在分子膜的两侧形成电势差，将成膜支架和电极3集成为一个整体，一起加工成型，形成生物芯片，进一步提高生物分子表征装置的结构紧凑性。优选的，在成膜腔处形成分子膜。
可以理解的是，储液腔13底部的电极3可以只与储液腔13内的导电液体导电连接，而通过生物芯片外部的电路与分子膜上侧的导电液体导电连接，设置二者的极性相反，实现分子膜两侧产生电势差。
另外，也可以设置电极3包括与储液腔13内的导电液体导电连接的部分，又包括与成膜腔14上部的液体导电连接的部分，两个部分的电极3的极性相反，同样可以实现在分子膜两侧产生电势差。
需要说明的是，电极3可以设置在绝缘基体2靠近第一绝缘层11的表面上，也可以嵌入在绝缘基体11结构内，这里不做限制。
第四方面，一种分子膜的制备方法，包括：
使用如上述的成膜支架、或如上方法制备得到的成膜支架，在成膜腔处形成分子膜。
该制备方法具体可以包括：
在成膜腔的表面预涂非极性试剂。需要说明的是，此时预涂的非极性试剂可以是用于形成分子膜的含有两亲性分子的非极性试剂，也可以是其他非极性试剂。预涂非极性试剂有利于提高气泡在生物芯片的表面上移动的附着性与顺畅性，提高分子膜的成膜效率。经过成膜腔向储液腔内加入 第一极性溶剂，直至第一极性溶剂的液面位于成膜腔内；向成膜腔内加入用于形成分子膜的非极性溶剂，非极性试剂可以是用于形成分子膜的含有两亲性分子的非极性试剂；向成膜腔内加入第二极性溶剂，以使非极性溶剂在成膜腔内形成分子膜。第一极性溶剂和第二极性溶剂可以相同，也可以不同。在加入非极性溶剂时，非极性溶剂会在第一极性溶剂的表面自由扩散开来，但是，此时经自由扩散的非极性溶剂还比较厚，且在第一极性溶剂的表面分布的还不够均匀。通过向成膜腔内加入第二极性溶剂，可以促使非极性溶剂在第一极性溶剂的上表面继续扩散，形成较薄的分子膜，即完成“油水互赶”的过程。
可以理解的是，需要综合考虑成膜腔的截面大小和需要形成的分子膜的厚度，对加入成膜腔内用于形成分子膜的非极性溶剂定量，或者调整非极性溶剂的组分。
需要说明的是，本申请实施例提供的成膜支架、生物芯片以及纳米孔表征生物分子的装置，在使用过程中，并不限于第四方面提供的成膜方法，也可以采用其它方法制备成膜支架和分子膜。
第五方面，本申请实施例提供一种生物分子表征装置，包括以上任意一实施例提供的成膜支架，后者包括以上任意一实施例提供的生物芯片。
本申请实施例提供的生物分子表征装置，由于采用了以上任意一实施例提供的成膜支架，或者采用以上任意一实施例提供的生物芯片，因而具有相同的技术效果，这里不再赘述。
第六方面，本申请实施例提供一种以上任意一实施例提供的成膜支架，以上任意一实施例制备得到的成膜支架，以上任意一实施例提供的生物芯片，以上任意一实施例提供的生物分子表征装置在生物分子表征或制备用于生物分子表征的产品中的用途，生物分子包括生物聚合物，生物聚合物选自多核苷酸、多肽、多糖和脂质中的一种，可选为多核苷酸，多核苷酸包括DNA和/或RNA。
示例性地，在生物分子表征的应用中，由于采用了上述任意一实施例提供的成膜支架、生物芯片、生物分子表征装置，因而具有相同的技术效果，在此不再赘述。
另外，本文中术语“和/或”，仅仅是一种描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B这三种情况。另外，本文中字符“/”，一般表示前后关联对象是一种“或”的关系。
应理解，在本申请实施例中，“与A相应的B”表示B与A相关联，根据A可以确定B。但还应理解，根据A确定B并不意味着仅仅根据A确定B，还可以根据A和/或其它信息确定B。
以上，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到各种等效的修改或替换，这些修改或替换都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以权利要求的保护范围为准。

Claims (20)

  1. 一种成膜支架，用于形成分子膜，其中，所述成膜支架包括：
    绝缘基体；和
    绝缘层，设置于所述绝缘基体上，所述绝缘层包括第一绝缘层和第二绝缘层，所述绝缘基体、所述第一绝缘层和所述第二绝缘层依次叠设，所述第一绝缘层中设置有储液腔，所述第二绝缘层中设置有与所述储液腔连通的成膜腔，所述成膜腔和所述储液腔朝向背离所述绝缘基体的方向开口，沿从所述绝缘基体至所述第二绝缘层的方向，所述成膜腔的开口尺寸逐渐增大。
  2. 根据权利要求1所述的成膜支架，其中，第一绝缘层的内表面构成所述储液腔的储液表面，所述第二绝缘层的内表面构成所述成膜腔的成膜表面，所述第二绝缘层背离所述绝缘基体的顶面与形成所述成膜腔的所述成膜表面平滑连接，所述成膜表面与形成所述储液腔的所述储液表面平滑连接。
  3. 根据权利要求2所述的成膜支架，其中，所述成膜表面为倾斜面或弧形面。
  4. 根据权利要求2所述的成膜支架，其中，所述第二绝缘层还包括自所述成膜表面凸出设置的支撑凸起。
  5. 根据权利要求4所述的成膜支架，其中，所述支撑凸起的数量为多个，多个所述支撑凸起沿从所述储液表面到所述顶面的方向依次设置。
  6. 根据权利要求5所述的成膜支架，其中，多个所述支撑凸起中的每个所述支撑凸起包括支撑沿和上升沿，所述上升沿沿从所述绝缘基体至所述第二绝缘层的方向延伸，所述支撑沿沿所述绝缘基体所在水平面延伸，沿从所述储液表面到所述顶面的方向，依次设置的各个所述支撑凸起的所 述支撑沿到所述绝缘基体的距离逐渐增加。
  7. 根据权利要求5所述的成膜支架，其中，多个所述支撑凸起中的每个所述支撑凸起包括下降沿和上升沿，所述下降沿设置于所述上升沿远离所述成膜腔的轴线的一侧，
    沿从所述储液表面到所述顶面的方向，依次设置的各个所述支撑凸起的所述上升沿的顶点到所述绝缘基体的距离逐渐增加。
  8. 根据权利要求5所述的成膜支架，其中，所述支撑凸起包括与所述顶面相连的多个依次连接的第一凸起，以及连接所述第一凸起和所述储液表面的第二凸起；沿从所述储液表面到所述顶面的方向，依次设置的各个所述第一凸起的顶点到所述绝缘基体的距离逐渐增加。
  9. 根据权利要求8所述的成膜支架，其中，所述第二凸起的数量为多个，多个所述第二凸起依次连接；沿从所述成膜腔的轴线至所述成膜腔的边沿方向，各个所述第二凸起的顶点在同一平面上。
  10. 根据权利要求4所述的成膜支架，其中，所述成膜支架还包括多个微凸起，多个所述微凸起至少设于所述成膜表面、所述储液表面和所述支撑凸起中的任一个。
  11. 根据权利要求1至10中任一项所述的成膜支架，其中，形成所述储液腔的储液表面沿从所述绝缘基体到所述第二绝缘层的方向设置。
  12. 根据权利要求1至10中任一项所述的成膜支架，其中，所述储液腔包括上腔室和下腔室，所述成膜腔、所述上腔室和所述下腔室依次连通，沿从所述绝缘基体到所述第二绝缘层的方向，所述上腔室的开口尺寸逐渐减小。
  13. 根据权利要求12所述的成膜支架，其中，所述上腔室的内壁面与所述绝缘基体所在平面之间的最大夹角为α，其中，1°＜α＜75°。
  14. 根据权利要求1至10中任一项所述的成膜支架，其中，多个所述储液腔呈阵列设置在所述第一绝缘层，多个所述成膜腔呈阵列设置在所述第二绝缘层，相邻所述成膜腔之间通过设置于所述第二绝缘层的第一通道连通；
    和/或，相邻所述储液腔之间通过设置于所述第一绝缘层的第二通道连通。
  15. 一种成膜支架的制备方法，其中，包括：
    提供绝缘基体，通过光刻工艺在所述绝缘基体上制备具有储液腔的第一绝缘层；
    在所述第一绝缘层远离所述绝缘基体的一侧制备固化的光致抗蚀层；
    通过具有预设图案的掩膜，对所述光致抗蚀层进行刻蚀，以在所述光致抗蚀层形成与所述储液腔连通的镂空区域，所述镂空区域为与所述预设图案对应的成膜腔，制备得到如权利要求1至14中任一项所述成膜支架。
  16. 一种成膜支架的制备方法，其中，包括：
    制备如权利要求1至14中任一项所述成膜支架对应反向结构的支架模板；
    对所述支架模板采用压印技术进行翻模转印，制备得到如权利要求1至14中任一项所述成膜支架。
  17. 一种生物芯片，其中，包括：
    如权利要求1至14中任一项所述的成膜支架或权利要求15至16中任一项所述方法制备得到的成膜支架；
    电极，设置于所述储液腔，所述电极能够与所述储液腔内的液体导电连接，且能够与所述生物芯片外部的电路连接。
  18. 一种分子膜的制备方法，其中，包括：
    使用权利要求1至14中任一项所述成膜支架、或权利要求15至16中任一项所述方法制备得到的成膜支架、或权利要求17所述的生物芯片，在所述成膜腔处形成分子膜。
  19. 一种纳米孔表征生物分子的装置，其中，包括如权利要求至1至14中任一项所述的成膜支架、如权利要求15至16中任一项所述方法制备得到的成膜支架、或者如权利要求17所述的生物芯片。
  20. 权利要求1至14中任一项所述的成膜支架、权利要求17所述的生物芯片、或权利要求19所述的纳米孔表征生物分子的装置在生物分子表征或制备用于生物分子表征的产品中的应用。
PCT/CN2022/132207 2022-01-10 2022-11-16 成膜支架、生物芯片、装置、制备方法及其应用 WO2023130839A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210022647.9 2022-01-10
CN202210022647.9A CN114460135A (zh) 2022-01-10 2022-01-10 成膜支架、生物芯片、装置、制备方法及其应用

Publications (1)

Publication Number Publication Date
WO2023130839A1 true WO2023130839A1 (zh) 2023-07-13

Family

ID=81409878

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/132207 WO2023130839A1 (zh) 2022-01-10 2022-11-16 成膜支架、生物芯片、装置、制备方法及其应用

Country Status (2)

Country Link
CN (1) CN114460135A (zh)
WO (1) WO2023130839A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114460135A (zh) * 2022-01-10 2022-05-10 成都齐碳科技有限公司 成膜支架、生物芯片、装置、制备方法及其应用
CN114751363B (zh) * 2022-06-14 2022-09-13 成都齐碳科技有限公司 孔阵列层结构、预涂方法、成膜方法及相关装置
WO2023240427A1 (zh) * 2022-06-14 2023-12-21 成都齐碳科技有限公司 孔阵列层结构、预涂方法、成膜方法及测序装置

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1460723A (zh) * 2002-05-15 2003-12-10 三星电子株式会社 具有亲水和疏水区域的生物分子芯片平板制备方法
CN104730137A (zh) * 2015-03-27 2015-06-24 中国科学院上海微系统与信息技术研究所 基于超薄绝缘层soi上的mosfet背栅生物传感器及其制备方法
JP2019146509A (ja) * 2018-02-26 2019-09-05 国立大学法人東北大学 シリコンチップ及びその製造方法
CN113145183A (zh) * 2020-01-22 2021-07-23 京东方科技集团股份有限公司 一种生物芯片及其制作方法
CN113426499A (zh) * 2021-07-08 2021-09-24 成都齐碳科技有限公司 微结构、生物芯片、成膜方法、基因测序装置及其应用
CN114460135A (zh) * 2022-01-10 2022-05-10 成都齐碳科技有限公司 成膜支架、生物芯片、装置、制备方法及其应用
CN216747520U (zh) * 2022-01-10 2022-06-14 成都齐碳科技有限公司 成膜支架、生物芯片和纳米孔表征生物分子的装置

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1460723A (zh) * 2002-05-15 2003-12-10 三星电子株式会社 具有亲水和疏水区域的生物分子芯片平板制备方法
CN104730137A (zh) * 2015-03-27 2015-06-24 中国科学院上海微系统与信息技术研究所 基于超薄绝缘层soi上的mosfet背栅生物传感器及其制备方法
JP2019146509A (ja) * 2018-02-26 2019-09-05 国立大学法人東北大学 シリコンチップ及びその製造方法
CN113145183A (zh) * 2020-01-22 2021-07-23 京东方科技集团股份有限公司 一种生物芯片及其制作方法
CN113426499A (zh) * 2021-07-08 2021-09-24 成都齐碳科技有限公司 微结构、生物芯片、成膜方法、基因测序装置及其应用
CN114460135A (zh) * 2022-01-10 2022-05-10 成都齐碳科技有限公司 成膜支架、生物芯片、装置、制备方法及其应用
CN216747520U (zh) * 2022-01-10 2022-06-14 成都齐碳科技有限公司 成膜支架、生物芯片和纳米孔表征生物分子的装置

Also Published As

Publication number Publication date
CN114460135A (zh) 2022-05-10

Similar Documents

Publication Publication Date Title
WO2023130839A1 (zh) 成膜支架、生物芯片、装置、制备方法及其应用
Zhang et al. Patterning colloidal crystals and nanostructure arrays by soft lithography
Yan et al. Microcontact printing of colloidal crystals
US10026609B2 (en) Nanoshape patterning techniques that allow high-speed and low-cost fabrication of nanoshape structures
JP4931330B2 (ja) マイクロ流体構造、特にバイオチップを製造する方法および該方法によって得られた構造
Xia et al. Non-photolithographic methods for fabrication of elastomeric stamps for use in microcontact printing
Duan et al. Fabrication of nanofluidic devices
CN103945930B (zh) 可编程的阵列
US10162162B2 (en) Microfluidic systems and methods for hydrodynamic microvortical cell rotation in live-cell computed tomography
JP4179166B2 (ja) 疎水性ゾーン装置
US6969489B2 (en) Micro array for high throughout screening
JP2003039399A (ja) 薄膜パターン形成用スタンプ
US20190011424A1 (en) Molecular analysis system with well array
CN216747520U (zh) 成膜支架、生物芯片和纳米孔表征生物分子的装置
Peng et al. Direct ink writing combined with metal-assisted chemical etching of microchannels for the microfluidic system applications
JP2005211857A (ja) 樹脂製マイクロチャネル基板及びその製造方法
Sethu et al. Cast epoxy-based microfluidic systems and their application in biotechnology
WO2020140732A1 (zh) 微流控基板及其制作方法和微流控芯片
WO2009125697A1 (ja) モールド、その製造方法および転写微細パターンを有する基材の製造方法
Mukherjee et al. Soft lithography meets self-organization: Some new developments in meso-patterning
Kang et al. Tightly sealed 3D lipid structure monolithically generated on transparent SU-8 microwell arrays for biosensor applications
JP2004154769A (ja) バイオミメティックメンブランの製造方法およびバイオミメティックメンブランならびにその応用
Lee et al. Replica mold for nanoimprint lithography from a novel hybrid resin
Jahn et al. Polymer microsieves manufactured by inkjet technology
Li et al. Facile fabrication of flexible polymeric membranes with micro and nano apertures over large areas

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22918291

Country of ref document: EP

Kind code of ref document: A1