CN114460135A

CN114460135A - Film-forming support, biochip, device, preparation method and application thereof

Info

Publication number: CN114460135A
Application number: CN202210022647.9A
Authority: CN
Inventors: 张喆; 夏晓翔; 宋璐
Original assignee: Chengdu Qitan Technology Ltd
Current assignee: Chengdu Qitan Technology Ltd
Priority date: 2022-01-10
Filing date: 2022-01-10
Publication date: 2022-05-10
Also published as: WO2023130839A1

Abstract

The invention discloses a film forming support, a biochip, a device, a preparation method and application thereof. The molecular membrane prepared by the membrane-forming bracket provided by the invention has good stability.

Description

Film-forming support, biochip, device, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a film-forming support, a preparation method of the film-forming support, a biochip, a preparation method of a molecular film, a device for representing biomolecules by using nanopores and application of the device.

Background

In the field of nanopore gene sequencing technology, it is generally necessary to form a molecular membrane on a biochip to embed a nanopore structure on the molecular membrane. When a biomolecule (e.g., a polynucleotide, a polypeptide, a polysaccharide, or a lipid) passes through a nanopore, the degree of current interruption due to analytes having different structures is different, so that sequence arrangement information or modification information of the biomolecule, etc. can be analytically obtained by measuring a change in an electric signal such as a current.

The existing molecular membrane is prepared by a bracket with a cavity, the membrane forming position of the molecular membrane in the cavity is mainly controlled by the position of a polar and non-polar medium layer interface, but the membrane forming position of the molecular membrane in the cavity is difficult to accurately control due to the medium layer interface being a liquid surface with a convex middle part and concave two sides, and the stability of the prepared molecular membrane is difficult to guarantee.

Disclosure of Invention

The embodiment of the invention provides a film-forming support, a preparation method of the film-forming support, a biochip, a preparation method of a molecular film, a device for representing biomolecules by using nanopores and application of the device, and aims to solve the problem of poor stability of the molecular film prepared by the conventional molecular film manufacturing equipment.

The first aspect of the embodiment of the invention provides a film forming support for forming a molecular film, which comprises an insulating base body and an insulating layer, wherein the insulating layer is arranged on the insulating base body, the insulating layer comprises a first insulating layer and a second insulating layer, the insulating base body, the first insulating layer and the second insulating layer are sequentially stacked, a liquid storage cavity is arranged in the first insulating layer, a film forming cavity communicated with the liquid storage cavity is arranged in the second insulating layer, the film forming cavity and the liquid storage cavity are opened towards a direction deviating from the insulating base body, and the opening size of the film forming cavity is gradually increased along a direction from the insulating base body to the second insulating layer.

Optionally, the inner surface of the first insulating layer forms a liquid storage surface of the liquid storage cavity, the inner surface of the second insulating layer forms a film forming surface of the film forming cavity, the top surface of the second insulating layer, which is away from the insulating substrate, is smoothly connected with the film forming surface forming the film forming cavity, and the film forming surface is smoothly connected with the liquid storage surface forming the liquid storage cavity.

Optionally, the film forming surface is an inclined surface or an arc surface.

Optionally, the second insulating layer further includes a support protrusion provided to protrude from the film formation surface.

Optionally, the number of the supporting protrusions is multiple, and the supporting protrusions are sequentially arranged in the direction from the liquid storage surface to the top surface.

Optionally, each of the plurality of supporting protrusions includes a supporting edge and a rising edge, the rising edge extends along a direction from the insulating base to the second insulating layer, the supporting edge extends along a horizontal plane where the insulating base is located, and a distance from the supporting edge to the insulating base of each supporting protrusion arranged in sequence increases gradually along a direction from the surface of the liquid storage to the top surface.

Optionally, each of the plurality of support protrusions includes a falling edge and a rising edge, the falling edge is disposed on one side of the rising edge away from the axis of the film forming chamber, and the distance from the vertex of the rising edge of each support protrusion disposed in sequence to the insulating base body gradually increases in the direction from the liquid storage surface to the top surface.

Optionally, the supporting protrusions include a plurality of first protrusions connected with the top surface in sequence, and second protrusions connecting the first protrusions and the liquid storage surface; the distance from the vertex of each first protrusion to the insulating substrate is gradually increased along the direction from the liquid storage surface to the top surface.

Optionally, the number of the second protrusions is multiple, and the multiple second protrusions are connected in sequence; the apexes of the respective second protrusions are on the same plane in the direction from the axis of the film-forming chamber to the rim of the film-forming chamber.

Optionally, the film-forming scaffold further comprises a plurality of microprotrusions provided on at least any one of the film-forming surface, the reservoir surface and the support protrusions.

Optionally, a liquid storage surface forming the liquid storage cavity is disposed in a direction from the insulating base to the second insulating layer.

Optionally, the liquid storage cavity comprises an upper cavity and a lower cavity, the film forming cavity, the upper cavity and the lower cavity are sequentially communicated, and the size of the opening of the upper cavity is gradually reduced along the direction from the insulating substrate to the second insulating layer.

Optionally, the maximum included angle between the inner wall surface of the upper cavity and the plane of the insulating base body is alpha,

wherein, the alpha is more than 1 degree and less than 75 degrees.

Optionally, the plurality of liquid storage cavities are arranged on the first insulating layer in an array mode, the plurality of film forming cavities are arranged on the second insulating layer in an array mode, and adjacent film forming cavities are communicated through a first channel arranged on the second insulating layer;

and/or the adjacent liquid storage cavities are communicated through a second channel arranged on the first insulating layer.

In a second aspect, an embodiment of the present invention further provides a method for preparing a film-forming scaffold, including:

providing an insulating substrate, and preparing a first insulating layer with a liquid storage cavity on the insulating substrate through a photoetching process;

preparing a cured photoresist layer on the side of the first insulating layer away from the insulating substrate;

and etching the photoresist layer through a mask with a preset pattern to form a hollow area communicated with the liquid storage cavity in the photoresist layer, wherein the hollow area is a film forming cavity corresponding to the preset pattern, and the film forming support is prepared.

In a third aspect, an embodiment of the present invention further provides a method for preparing a film-forming scaffold, including:

preparing a support template of the film-forming support corresponding to the reverse structure;

and (3) performing mold turning and transfer printing on the support template by adopting an imprinting technology to prepare the film-forming support.

In a fourth aspect, embodiments of the present invention further provide a biochip, including:

a film-forming scaffold as described above or a film-forming scaffold prepared as described above;

the electrode is arranged in the liquid storage cavity, can be in conductive connection with liquid in the liquid storage cavity and can be connected with a circuit outside the biochip.

In a fifth aspect, an embodiment of the present invention further provides a method for preparing a molecular film, including:

a molecular film is formed in the film forming cavity by using the film forming support, the film forming support prepared by the method, or the biochip.

In a sixth aspect, embodiments of the present invention further provide a biomolecule characterization device, including the membrane-forming scaffold, the membrane-forming scaffold prepared by the method, or the biochip.

In a seventh aspect, the embodiments of the present invention further provide an application of the above film-forming scaffold, the above biochip, and the biomolecule characterization device in biomolecule characterization or preparation of products for biomolecule characterization.

In the film-forming support, the preparation method of the film-forming support, the biochip, the preparation method of the molecular film and the device for representing biomolecules by the nano-pores and the application thereof, the second insulating layer can provide a certain supporting force for the molecular film in the vertical direction by arranging the film-forming cavity with the gradually increased opening size, so that the liquid flow in the film-forming cavity is avoided or reduced, the liquid surface shape is optimized, and the relative position of the molecular film and the film-forming support is influenced; the contact area between the molecular film in the film forming cavity and the second insulating layer can be increased by preparing the film forming cavity into a structure with the opening size gradually increased; the second insulating layer can provide a supporting force for the molecular film, and an oil-water interface formed by the polar solvent and the nonpolar solvent is maintained, so that the position where the molecular film is formed can be accurately fixed.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a schematic cross-sectional view of a film-forming stent according to an embodiment of the present invention;

FIG. 2 is an enlarged structural view of portion A shown in FIG. 1;

FIG. 3 is a schematic cross-sectional view of a portion of a film-forming support according to an embodiment of the present invention;

FIG. 4 is a schematic cross-sectional view of a portion of a film-forming stent according to another embodiment of the present invention;

FIG. 5 is an enlarged view of a portion of the stent shown in FIG. 4;

FIG. 6 is a schematic sectional view of a portion of a film-forming stent according to yet another embodiment of the present invention;

FIG. 7 is an enlarged view of a portion of the stent shown in FIG. 6;

FIG. 8 is a schematic sectional view of a film-forming support according to still another embodiment of the present invention;

FIG. 9 is a schematic cross-sectional view of a portion of a film-forming stent according to an embodiment of the present invention;

FIG. 10 is a schematic cross-sectional view of a film-forming stent according to another embodiment of the present invention;

FIG. 11 is a schematic cross-sectional view of a stent according to another embodiment of the present invention;

FIG. 12 is a schematic cross-sectional view of a film-forming stent according to still another embodiment of the present invention;

FIG. 13 is a schematic flow chart of a method for manufacturing a stent according to an embodiment of the present invention;

FIG. 14 is a schematic view of a portion of a film-forming stent during fabrication according to an embodiment of the present invention;

FIG. 15 is a schematic view of a portion of a film-forming stent during fabrication according to another embodiment of the present invention;

FIG. 16 is a schematic structural diagram illustrating a process for manufacturing a stent according to still another embodiment of the present invention;

FIG. 17 is a schematic structural view of a scanning electron microscope of a film forming chamber according to an embodiment of the present invention;

FIG. 18 is a schematic structural view of a scanning electron microscope of a film forming chamber according to another embodiment of the present invention.

Description of reference numerals:

10. a film-forming scaffold; 1. an insulating layer; 11. a first insulating layer; 111. a surface of the stock solution; 12. a second insulating layer; 121. a top surface; 123. a film forming surface; 124. a support boss; 1241. a first protrusion; 1242. a second protrusion; 1243. a support edge; 1244. a rising edge; 1245. a falling edge; 125. micro-protrusions; 13. a liquid storage cavity; 131. an upper chamber; 132. a lower chamber; 14. a film forming cavity; 2. an insulating substrate; 3. an electrode;

20. photoresist; 30. a liquid storage cavity mask plate; 40. a photoresist layer; 50. photoresist; 501. a forward structure; 502. a planar pattern; 60. and (5) photoetching a mask plate.

Detailed Description

Features and exemplary embodiments of various aspects of the present invention will be described in detail below. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the present invention by illustrating examples of the present invention.

The directional terms used in the description of the invention are used for convenience in describing the invention and for simplicity in description, and do not indicate or imply that the apparatus or component being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the invention.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The embodiments will be described in detail below with reference to the accompanying drawings.

In a first aspect, embodiments of the present disclosure provide a film-forming stent. As shown in fig. 1 and 2, the film-forming support 10 includes an insulating substrate 2 and an insulating layer 1, the insulating layer 1 is disposed on the insulating substrate 2, the insulating layer 1 includes a first insulating layer 11 and a second insulating layer 12, the insulating substrate 2, the first insulating layer 11 and the second insulating layer 12 are sequentially stacked, and a liquid storage cavity 13 is disposed in the first insulating layer 11; be provided with the film forming cavity 14 with stock solution chamber 13 intercommunication in the second insulating layer 12, film forming cavity 14 and stock solution chamber 13 orientation are kept away from insulating base member 2's direction opening, along the direction from insulating base member 2 to second insulating layer 12, and film forming cavity 14's opening size increases gradually.

The film-forming support 10 is used for preparing a molecular film, specifically, the molecular film can be a molecular film with an amphiphilic molecular layer, a plurality of nanopore structures communicated with two sides of the molecular film are arranged in the amphiphilic molecular layer, and the molecular film can be applied to biomolecule characterization based on a nanopore sequencing technology. The biomolecule may specifically be one of a polynucleotide, a polypeptide, a polysaccharide and a lipid, the polynucleotide comprising DNA and/or RNA.

The film forming chamber 14 formed on the film forming support 10 is a chamber having an opening through which a reagent for preparing a molecular film can be added into the film forming chamber 14 or into the liquid storage chamber 13 through the film forming chamber 14. It can be understood that the insulating substrate 2, the first insulating layer 11 and the second insulating layer 12 are sequentially arranged along the vertical direction, and the opening is arranged opposite to the insulating substrate 2, so that the preparation and the use of the film-forming support 10 are facilitated. The reservoir 13 can contain a polar solvent, such as a buffer solution for preparing a molecular membrane, specifically a phosphate buffer solution, a HEPES buffer solution containing KCl or NaCl, a CAPS buffer solution containing KCl or NaCl, or the like. The liquid level of the polar solvent in the liquid storage cavity 13 is controlled, 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 cavity 14, the non-polar solvent can be a non-polar solvent for dissolving the amphiphilic material, such as silicone oil, specifically methyl phenyl silicone oil, polydimethylsiloxane and the like, and the polar solvent is driven, so that the process of oil driving and water driving is completed. On the basis, polar solvent is introduced to drive the nonpolar solvent of the amphiphilic material, and the process of 'water oil drive' is completed. At this time, a structure of polar solvent-nonpolar solvent-polar solvent may be formed in the film forming chamber 14, and the nonpolar solvent of the amphiphilic material is sandwiched between two layers of polar solvents to form a molecular film. The molecular film is partially suspended in the film forming chamber 14 and partially attached to the wall surface 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 cavity 14 and the liquid storage cavity 13 are not limited, and only the liquid storage cavity 13 can be ensured to contain polar solvent, and a molecular film can be formed in the film forming cavity 14. The size and shape of the opening are not limited, as long as a molecular film can be formed to meet the requirement of subsequent characterization of biomolecules.

After the molecular membrane with the transmembrane nano-pores is prepared on the membrane-forming bracket 10, a certain electric potential can be applied, an analyte passes through the transmembrane nano-pores under the action of the electric potential and causes current or voltage change, and the characterization information of the analyte can be obtained according to the current or voltage change information. For example, size information, sequence information, identity information, modification information, etc. of the analyte are obtained according to the current change information. The high-quality molecular film prepared by the film-forming support 10 is particularly important for the accuracy and the timeliness of the subsequent characterization of the biomolecules.

At least two of the insulating substrate 2, the first insulating layer 11 and the second insulating layer 12 are made of the same material, so that the preparation is convenient. Of course, three different materials may be used for the respective preparation. The first insulating layer 11 and the second insulating layer 12 may be sequentially formed on the insulating base 2 by a deposition process, such as a chemical vapor deposition or a plasma enhanced deposition process. It will be appreciated that the insulating base 2, the first insulating layer 11, and the second insulating layer 12 may all be made of a dielectric material, such as silicon dioxide. It can be understood that, the nanopore sequencing technology needs to form a potential difference on two sides of the molecular membrane, the electrodes 3 may be further disposed so as to form a potential difference on two sides of the molecular membrane, and the electrodes 3 may be locally localized on the membrane-forming support 10 by combining processes such as metal deposition and/or electroplating, so that the molecular membrane prepared by the membrane-forming support 10 has the possibility of electrical characterization test and use in the environment of polar liquid.

The inner surface of the first insulating layer 11 forms a liquid storage surface 111 of the liquid storage cavity 13, the inner surface of the second insulating layer 12 forms a film forming surface 123 of the film forming cavity 14, and the film forming surface 123 can be an inclined surface, an arc surface or a surface with structures such as supporting steps, protrusions and the like; by providing the film-forming chamber 14 with a structure in which the opening size is gradually increased, the contact area of the molecular film in the film-forming chamber 14 with the first insulating layer 11 can be increased; since the first insulating layer 11 can provide a supporting force to the molecular film, an oil-water interface formed by the polar solvent and the non-polar solvent, that is, a position where the molecular film is formed, can be accurately fixed.

As shown in fig. 1 and 2, the top surface 121 of the second insulating layer 12 facing away from the insulating substrate 2 is smoothly connected to the film formation surface 123 forming the film formation cavity 14, and the film formation surface 123 is smoothly connected to the liquid storage surface 111 forming the liquid storage cavity 13. The top surface 121 may be a flat surface or a curved surface having a certain curvature. By arranging smooth connection among the surfaces, the prepared molecular membrane is prevented from being punctured by a sharp connecting angle, and the stability of preparing the molecular membrane is improved.

In one embodiment, the film formation surface 123 is an inclined surface, i.e., the plane of the film formation surface 123 intersects the direction perpendicular to the insulating substrate 2 at an angle, and the film formation cavity 14 formed by the film formation surface 123 is in the shape of a bell mouth. In another embodiment, as shown in FIG. 3, the film forming surface 123 is an arcuate surface. The arc-shaped surface can be a concave surface as shown in fig. 3, and can also be a convex surface, and a person skilled in the art can select a proper type and arc degree of the arc-shaped surface according to needs. No matter the film forming surface 123 is an inclined surface or an arc surface, a certain supporting force can be provided for the molecular film, and meanwhile, the flat inclined surface and the flat arc surface can avoid puncturing the molecular film, so that the stability of the molecular film is improved.

In another embodiment, as shown in fig. 4, the second insulating layer 12 further includes a support protrusion 124 provided to protrude from the film formation surface 123. The supporting protrusion 124 can obstruct the flow of liquid in the cavity, thereby improving the stability of the prepared molecular membrane; meanwhile, the prepared molecular film can be completely attached to the whole support protrusion 124, so that the contact area between the molecular film and the second insulating layer 12 is increased, and the molecular film can be fixed by the second insulating layer 12.

The number of the supporting protrusions 124 may be plural, and the plural supporting protrusions 124 are sequentially arranged in a direction from the liquid storage surface 111 to the top surface 121. The specific shape of each support protrusion 124 is not limited, and the shape of the support protrusions 124 arranged in sequence may be uniform or non-uniform, but the surface of each support protrusion 124 is a curved surface.

As shown in fig. 4 and 5, in an embodiment, each of the plurality of supporting protrusions 124 includes a supporting edge 1243 and a rising edge 1244, the rising edge 1244 extends along a direction from the insulating substrate 2 to the second insulating layer 12, the supporting edge 1243 extends along a horizontal plane of the insulating substrate 2, and a distance from the supporting edge 1243 to the insulating substrate 2 of each supporting protrusion 124 sequentially increases along a direction from the liquid storage surface 111 to the top surface 121.

As shown in fig. 6 and 7, in another embodiment, each of the plurality of support protrusions 124 includes a falling edge 1245 and a rising edge 1244, the falling edge 1245 is disposed on a side of the rising edge 1244 away from the axis x of the film-forming chamber 14, and the distance from the apex of the rising edge 1244 of each support protrusion 124 disposed in sequence to the insulating base 2 gradually increases in the direction from the liquid storage surface 111 to the top surface 121. The axis x of the film-forming chamber 14 is arranged in 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. The different support projections 124 are connected in a plane parallel to the insulating base body 2, and an annular projection provided along the film-forming chamber 14 may be formed, and the different support projections 124 are connected in different planes, and a plurality of annular projections may be formed.

Referring to fig. 8, the supporting bump 124 includes a plurality of first bumps 1241 connected to the top surface 121 in sequence, and a second bump 1242 connecting the first bumps 1241 and the liquid storage surface 111, where the first bumps 1241 are connected to the top surface 121 of the second insulating layer 12; the distance from the apex of each of the first protrusions 1241 arranged in sequence to the insulating base 2 gradually increases in the direction from the liquid storage surface 111 to the top surface 121. In this embodiment, the vertexes of the plurality of first protrusions 1241 are disposed along a slope such that the corresponding first protrusion 1241 can support and fix the molecular membrane floating on the polar solvent no matter which first protrusion 1241 the liquid surface of the polar solvent corresponds to, and the aforementioned "corresponding" means that the first protrusion 1241 contacts with the edge of the liquid surface of the polar solvent.

Further, the number of the second protrusions 1242 is plural, and the plural second protrusions 1242 are connected in sequence; the apexes of the respective second protrusions 1242 in the direction from the axis x of the film-forming chamber 14 to the rim of the film-forming chamber 14 are 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 may restrict liquid fluctuation, thereby improving stability of the molecular film.

Referring to fig. 9, the stent 10 further includes micro-protrusions 125, and the micro-protrusions 125 are disposed on at least one of the film-forming surface 123, the liquid-storing surface 111, and the supporting protrusions 124. The micro-protrusions 125 may be sequentially spaced apart, or may be irregularly arranged. Optionally, the interval between adjacent micro-protrusions 125 is 0.1-10 μm, and the size of a single micro-protrusion 125 is 5 nm-50 nm. The micro-protrusions 125 may be in the form of dots with semicircular protrusions, cylinders with a certain length, tentacles, and the like. The micro-protrusions 125 may be simultaneously disposed on the film-forming surface 123, the support protrusions 124, and the reservoir surface 111. By providing the micro-protrusions 125, the liquid fluctuation is further restricted, thereby improving the stability of the molecular membrane.

Further, the liquid storage surface 111 forming the liquid storage chamber 13 is disposed in a direction from the insulating substrate 2 to the second insulating layer 12, i.e., the liquid storage surface 111 may be disposed perpendicular to the insulating substrate 2. The cavity surrounded by the film-forming surface 123 and the liquid-storing surface 111 may or may not have a uniform shape when viewed in a direction perpendicular to the insulating substrate 2. In some embodiments, the cross section of the film-forming chamber 14 surrounded by the film-forming surfaces 123 in the direction perpendicular to the insulating base 2 has an obtuse angle, an acute angle, or a right angle, and specifically, may be formed with any one or more of an obtuse angle, an acute angle, or a right angle. It can be understood that the molecular film formed by the polar solvent has a tendency of gathering with an angled inner wall in the film forming cavity 14, so that the region can relatively stably pull the molecular film, and has a relatively stable supporting effect on the molecular film, and the molecular film formed in this way is more stable and has a higher film forming rate. The cross section of the film forming chamber 14 may be circular or elliptical, or may be polygonal such as triangular, rectangular, pentagonal, or polygonal. It is understood that the above cross-section has a centrosymmetric structure, which can improve the supporting effect of the film formation surface 123 on the molecular film.

It is understood that the shape of the reservoir 13 is not limited and may be polygonal or irregular. Illustratively, the reservoir 13 is in the shape of a polygonal prism or an elliptic cylinder, optionally a cylinder. When the shape of the liquid storage cavity 13 is cylindrical, the processing of the film-forming bracket 10 is convenient.

The cross section of the film forming cavity 14 can be larger than, equal to or smaller than that of the liquid storage cavity 13, namely, the orthographic projection of the film forming cavity 14 towards the liquid storage cavity 13 covers the liquid storage cavity 13, or the edge is overlapped with the edge of the liquid storage cavity 13, or the film forming cavity falls into the liquid storage cavity 13. In some embodiments, the orthographic projection of the film forming cavity 14 to the direction of the liquid storage cavity 13 coincides with the liquid storage cavity 13, that is, the cross section of the film forming cavity 14 is equal to that of the liquid storage cavity 13, so that the liquid storage cavity 13 and the film forming cavity 14 are more conveniently prepared, the yield and the consistency of the film forming support 10 are improved, and the film forming stability and the film forming rate of the molecular film in the film forming cavity 14 are improved. The liquid storage cavity 13 can be prepared into a structure with the size being sequentially reduced or gradually enlarged along the direction from the film forming cavity 14 to the liquid storage cavity 13 according to the factors such as the storage capacity, the film forming size and the like of the liquid storage cavity 13 required by a user.

Referring to fig. 10, in an embodiment, the liquid storage chamber 13 includes an upper chamber 131 and a lower chamber 132, the film forming chamber 14, the upper chamber 131 and the lower chamber 132 are sequentially communicated, and the opening size of the upper chamber 131 gradually decreases along a direction from the insulating substrate 2 to the second insulating layer 12. Thereby having larger liquid storage capacity, and simultaneously, the opening size at the connection part with the film forming cavity 14 is smaller, so that the exposed size of the film forming liquid surface is smaller, and the liquid surface movement is reduced. Of course, the extension direction of the liquid storage surface 111 can be varied, and different included angles are formed between the liquid storage surface and the plane where the insulating substrate 2 is located, so that liquid storage cavities 13 with different structures are formed, and different requirements are met. The plane of the insulating substrate 2 is taken as a horizontal plane, and the maximum included angle between the inner wall surface of the upper chamber 131 and the horizontal plane is alpha, wherein 1 degree < alpha < 75 degrees, preferably 5 degrees < alpha < 60 degrees, and more preferably 15 degrees < alpha < 55 degrees. The maximum included angle beta between the film formation surface 123 and the horizontal plane is 10 to 80 degrees, preferably 30 to 60 degrees, or the maximum included angle beta between the line connecting the vertexes of two adjacent support protrusions 124 and the horizontal plane is 10 to 80 degrees, preferably 30 to 60 degrees.

In the sectional structure of the liquid storage chamber 13 in the direction from the insulating base 2 to the second insulating layer 12, the inner wall surface of the upper chamber 131 may be in a linear state as shown in fig. 10; as shown in fig. 11, the insulating substrate 2 may be curved so as to protrude toward the insulating substrate; or as shown in fig. 12, it is in a curved state protruding in a direction away from the insulating base 2. That is, the inner wall surface of the upper chamber 131 may be a tilted plane or a curved surface. Referring to fig. 11 and 12, a dotted line is a tangent line of a certain point of the inner wall surface of the upper chamber 131. When the inner wall surface is a curved surface, each point of the curved surface is taken as a tangent, and the maximum included angle between each tangent and the plane where the lining insulation matrix 2 is located is alpha. That is, the included angle α in the embodiment of the present application means that the maximum included angle formed between the tangent plane of the inner wall surface and the plane of the substrate is the included angle α no matter the inner wall surface forming the upper chamber 131 is a plane or a curved surface. The inner wall surface of the upper cavity 131 is set to be an inclined plane or a curved surface, so that the polar liquid in the liquid storage cavity 13 can be maintained to have a certain shape, the stability of the polar liquid in the liquid storage cavity 13 is facilitated, and the product transportation and long-term storage are facilitated.

It is understood that one liquid storage cavity 13 may be disposed in the first insulating layer 11, and one film forming cavity 14 may be correspondingly disposed in the second insulating layer 12, or a plurality of liquid storage cavities 13 may be disposed in the first insulating layer 11, and a plurality of film forming cavities 14 may be correspondingly disposed in the second insulating layer 12, which is not limited herein and is selected according to specific requirements. When a plurality of liquid storage cavities 13 and a plurality of film forming cavities 14 are arranged on one film forming support 10, the plurality of liquid storage cavities 13 can be regularly arranged on the first insulating layer 11 in an array mode, and the plurality of film forming cavities 14 can be regularly arranged on the second insulating layer 12 in an array mode, so that the number of the film forming cavities 14 and the number of the liquid storage cavities 13 distributed in a unit area are increased. Of course, there may be no regular arrangement, and no limitation is made here. In this way, one membrane-forming stent 10 can simultaneously prepare a plurality of molecular membranes to perform characterization of a plurality of biomolecules, thereby improving the efficiency of detection.

Optionally, the adjacent film forming cavities 14 are communicated through the first channel, that is, ions or molecules in the liquid contained in the film forming cavities 14 can diffuse through the channel, which is beneficial to maintaining the balance of the liquid concentration in each film forming cavity 14 and is convenient for adding solution into the film forming support 10. Similarly, the adjacent liquid storage cavities 13 can be communicated through the second channel, and ions or molecules in the liquid contained in the liquid storage cavities 13 can be diffused through the second channel, so that the balance of the liquid concentration in each liquid storage cavity 13 is kept.

In one embodiment, at least one of the adjacent liquid storage chambers 13 and the adjacent film forming chambers 14 are isolated from each other. Namely, the liquid storage cavities 13 of the same film forming support 10 are not communicated with each other, and the film forming cavities 14 of the same film forming support 10 are not communicated with each other, so that the independence of the sequencing work of the liquid storage cavities 13 and the corresponding film forming cavities 14 is favorably kept, and the mutual interference is avoided.

Referring to fig. 13, in a second aspect, an embodiment of the invention provides a method for preparing a film-forming scaffold, including:

s1, providing an insulating substrate, and preparing a first insulating layer with a liquid storage cavity on the insulating substrate through a photoetching process;

s2, preparing a cured photoresist layer on the side, away from the insulating substrate, of the first insulating layer;

and S3, etching the photoresist layer through a mask with a preset pattern, for example, performing dry etching or laser etching, so as to form a hollow area communicated with the liquid storage cavity in the photoresist layer, and preparing the film forming support, wherein the hollow area is a film forming cavity corresponding to the preset pattern.

Specifically, before S1, the method further includes: the insulating base body is provided with blind holes through a photoetching process, and electrodes are arranged in the blind holes through a metal deposition process, so that the electrodes can be distributed on the insulating base body. Referring to fig. 14(a) and 14(b), a photoresist 20 and/or a dry film is coated on the insulating substrate 2 and the electrodes 3; referring to fig. 14(c) and 14(d), the photoresist and/or the dry film is patterned by a photolithography process using a liquid storage cavity mask 30 to form a first insulating layer 11 having a liquid storage cavity 13, and the position of the liquid storage cavity 13 corresponds to the electrode 3, so that the electrode 3 can contact the solution contained in the liquid storage cavity 13. The ratio of the height of the liquid storage cavity 13 to the diameter of the liquid storage cavity 13 is (0.8-3): 1.

referring to fig. 15(a), a photoresist material is coated on an end of the first insulating layer 11 away from the insulating substrate 2, and cured after light irradiation to form a solid photoresist layer 40. The photoresist layer 40 is made of a photoresist material, which means that after a polymer material is irradiated by light, the molecular structure is changed from linear solubility to bodily insolubility, thereby generating the corrosion resistance to a solvent. The photoresist material may be a dichromate photoresist, a polyvinyl alcohol cinnamate photoresist, or the like.

In S3, the preset pattern may be a 2D pattern distributed on the mask, or may be a 3D pattern having a three-dimensional structure. The mask may be a reusable mask prepared in advance, or may be a mask layer formed by coating a photoresist layer.

In one embodiment, the predetermined pattern is a 3D pattern having a three-dimensional structure. S3 may include:

s31, coating a layer of photoresist on the photoresist layer, and preparing the photoresist into a mask layer with a forward structure by utilizing a gray scale photoetching process;

and S32, taking the mask layer with the positive structure as a mask, and etching the photoresist layer to form a hollow area corresponding to the positive structure on the photoresist layer, thereby preparing the film-forming support.

The forward structure is a 3D pattern designed in advance by the skilled person according to the structure of the film forming cavity. Referring to fig. 15(b), 15(c) and 15(D1), the photoresist 50 in S31 may be a positive photoresist, and at this time, the exposure rate is different at different positions in the photoresist 50 by using the mask 60 through a gray scale lithography process, so as to form a 3D pattern, i.e., the forward structure 501, on the photoresist 50.

Referring to fig. 15(e), the photoresist layer 40 having the forward structure 501 may be used as a mask to perform anisotropic etching and/or isotropic etching on the photoresist layer 40, so that the photoresist layer 40 has a hollow area corresponding to the forward structure 501, and the hollow area is the film forming cavity 14 to be prepared, that is, the film forming support having the film forming cavity 14 is prepared. Anisotropic etching is an etching method that exhibits different etching rates in different crystallographic planes, and isotropic etching generally refers to an etching method in which different crystallographic planes exhibit the same etching rate. One skilled in the art can choose different etching methods to process the photoresist layer 40 into the second insulating layer 12 with the film forming cavity 14 as required.

In another embodiment, the preset pattern may specifically be a 2D pattern distributed on the mask, and S3 includes:

s33, coating a layer of photoresist on the photoresist layer, and preparing the photoresist into a mask layer with a plane pattern through dry reactive etching;

and S34, taking the mask layer with the plane pattern as a mask, etching the photoresist layer to enable the photoresist layer to form a hollow area corresponding to the plane pattern, and preparing the film forming support.

Referring to fig. 15(d2) and fig. 15(e), in the above embodiment, the photoresist layer 40 is etched by using the photoresist 50 having the two-dimensional planar pattern 502 as a mask, so as to process a hollow area on the photoresist layer 40 according to the planar pattern 502 on the photoresist 50, the etching selection ratio between materials, and the like, thereby preparing the above-mentioned film-forming support.

Unlike S32, the mask layer used to process the photoresist layer 40 in S34 may be a two-dimensional planar patterned photoresist and/or a patterned inorganic material film layer.

In yet another embodiment, S3 includes:

s35, preparing a mask with a preset pattern, and transferring the preset pattern to the photoresist layer through laser etching to prepare the film-forming support. The laser etching is preferably excimer laser etching.

Specifically, the photoresist layer may specifically be an organic high molecular polymer such as: epoxy resins, trimethacrylates, imides, polycarbonates, and the like; the mask can be a quartz mask chromium plate, a metal hollow mask and the like. When excimer laser etching is carried out, a mask can be placed between an excimer laser and a photoresist layer, ultraviolet photons are radiated by the laser, and the photoresist layer is partially decomposed to form the film-forming support with a required structure.

Referring to FIG. 16, the present application also provides another method for preparing a film-forming scaffold, comprising:

s4, preparing a support template of the film-forming support corresponding to the reverse structure;

and S5, performing mold turning and transfer printing on the support template by adopting an imprinting technology to prepare the film-forming support structure.

The support template is a reverse structure mold prepared in advance according to a structure required by the film-forming support, and the support template may be a metal mold specifically formed by electroforming. Referring to fig. 16(a), when performing the transfer printing by the imprinting technique, the insulating substrate 2 having the circuit structure may be prepared in advance; referring to fig. 16(b) and 16(c) and 16(d), an imprint glue 60 is coated on the insulating substrate 2, the thickness of the imprint glue 60 is the same as the sum of the thicknesses of the first insulating layer 11 and the second insulating layer 12 of the stent to be formed, the stent template 70 is pressed on the surface of the substrate, and the inverse structure of the stent template 70 is reversely transferred onto the imprint glue 60 by means of pressing; referring to fig. 16(e), a film-formed stent having a forward structure, i.e., a film-formed stent having a first insulating layer 11 and a second insulating layer 12, is prepared. S5 may be followed by: the residual imprint resist 60 is removed by a dry etching method to improve the conduction performance and the processing precision of the bottom electrode. Of course, the removed photoresist can be etched using an excimer laser. It can be understood that, in some embodiments, because the film forming cavity or the liquid storage cavity has a complex structure, which causes difficulty in demolding during the mold-flipping transfer process, a plurality of sub-templates with different structures can be prepared according to the structure of the film forming support to be prepared, then the sub-templates are subjected to mold-flipping transfer to obtain a plurality of parts, and the parts are bonded to obtain the film forming support. For example, a first sub-template having a reverse structure corresponding to the first insulating layer and a second sub-template having a reverse structure corresponding to the second insulating layer are prepared in advance, and then the first sub-template and the second sub-template are subjected to mold turning and transfer printing to obtain a first insulating layer and a second insulating layer, and the insulating substrate, the first insulating layer and the second insulating layer are sequentially bonded to obtain the film-forming support.

The manner of preparing the film-forming stent is not limited to the above-listed manner, and the film-forming stent prepared by the above-mentioned method has the same technical effects, and thus the detailed description thereof is omitted.

Referring to fig. 17 and 18, the schematic structural view of a scanning electron microscope for preparing a film forming cavity by a photolithography process according to the present invention is shown, 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 has an annular protrusion formed by a second protrusion 1242 and a plurality of first protrusions 1241 connected in sequence.

In a third aspect, an embodiment of the present invention provides a biochip, as shown in fig. 1, the biochip includes an electrode 3 and the film-forming support provided in any of the above embodiments, the electrode 3 is disposed at the bottom of the liquid storage cavity 13, and can be electrically connected to the liquid in the liquid storage cavity 13 and can be connected to a circuit outside the biochip. The electrode 3 is used for forming potential difference on two sides of the molecular membrane, and the membrane forming support and the electrode 3 are integrated into a whole and are processed and molded together to form a biological chip, so that the structural compactness of the biological molecule characterization device is further improved. Preferably, a molecular film is formed at the film-forming chamber.

It can be understood that the electrode 3 at the bottom of the liquid storage cavity 13 can be electrically connected with the conductive liquid in the liquid storage cavity 13 only, and is electrically connected with the conductive liquid at the upper side of the molecular membrane through a circuit outside the biochip, and the polarities of the two are set to be opposite, so that the potential difference is generated at the two sides of the molecular membrane.

In addition, the electrode 3 can also be provided with a part which is electrically connected with the conductive liquid in the liquid storage cavity 13 and a part which is electrically connected with the liquid at the upper part of the film forming cavity 14, the polarity of the electrodes 3 of the two parts is opposite, and the generation of the potential difference at the two sides of the molecular film can also be realized.

It should be noted that the electrode 3 may be disposed on the surface of the insulating base 2 close to the first insulating layer 11, or may be embedded in the insulating base 11, which is not limited herein.

In a fourth aspect, a method of preparing a molecular membrane comprises:

using the film-forming scaffold as described above, or a film-forming scaffold prepared as described above, a molecular film is formed at the film-forming cavity.

The preparation method specifically comprises the following steps:

the surface of the film forming cavity is pre-coated with a non-polar reagent. The nonpolar reagent to be precoated may be a nonpolar reagent containing an amphiphilic molecule for forming a molecular membrane, or may be another nonpolar reagent. Pre-coating the non-polar reagent is favorable to raising the adhesion and smoothness of bubble moving on the surface of biochip and raising the filming efficiency of molecular film. Adding a first polar solvent into the liquid storage cavity through the film forming cavity until the liquid level of the first polar solvent is positioned in the film forming cavity; adding a nonpolar solvent for forming the molecular membrane into the membrane forming cavity, wherein the nonpolar solvent can be a nonpolar agent containing amphipathic molecules for forming the molecular membrane; and adding a second polar solvent into the film forming cavity, so that the nonpolar 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. When the nonpolar solvent is added, the nonpolar solvent can freely diffuse out on the surface of the first polar solvent, but the freely diffused nonpolar solvent is still thicker and not uniformly distributed on the surface of the first polar solvent. By adding the second polar solvent into the film forming cavity, the non-polar solvent can be promoted to continuously diffuse on the upper surface of the first polar solvent to form a thinner molecular film, namely the process of mutual oil-water driving is completed.

It is understood that the non-polar solvent to be added into the film-forming chamber for forming the molecular film is quantified or the composition of the non-polar solvent is adjusted in consideration of the sectional size of the film-forming chamber and the thickness of the molecular film to be formed.

In the application, the film-forming support, the biochip and the device for characterizing a biomolecule by a nanopore provided in the embodiment of the present application are not limited to the film-forming method provided in the fourth aspect, and other methods may be used to prepare the film-forming support and the molecular membrane.

In a fifth aspect, embodiments of the present invention provide a biomolecule characterization device, including a membrane-forming scaffold provided in any one of the above embodiments, the membrane-forming scaffold including a biochip provided in any one of the above embodiments.

The biomolecule characterization device provided by the embodiment of the invention has the same technical effect due to the adoption of the film-forming support provided by any one of the above embodiments or the adoption of the biochip provided by any one of the above embodiments, and the details are not repeated herein.

In a sixth aspect, embodiments of the present invention provide a film-forming scaffold provided in any one of the above embodiments, a film-forming scaffold prepared in any one of the above embodiments, a biochip provided in any one of the above embodiments, and use of a biomolecule characterization device provided in any one of the above embodiments in biomolecule characterization or preparation of a product for biomolecule characterization, a biomolecule including a biopolymer, the biopolymer being selected from one of a polynucleotide, a polypeptide, a polysaccharide and a lipid, optionally a polynucleotide, the polynucleotide including DNA and/or RNA.

For example, in the application of biomolecule characterization, since the film-forming support, the biochip and the biomolecule characterization device provided in any of the above embodiments are used, the same technical effects are obtained, and no further description is provided herein.

In addition, the term "and/or" herein is only one kind of association relationship describing an associated object, and means that there may be three kinds of relationships, for example, a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. In addition, the character "/" herein generally indicates that the former and latter related objects are in an "or" relationship.

It should be understood that in the embodiment of the present invention, "B corresponding to a" means that B is associated with a, from which B can be determined. It should also be understood that determining B from a does not mean determining B from a alone, but may be determined from a and/or other information.

While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A film-forming scaffold for forming a molecular film, comprising:

an insulating substrate; and

the insulating layer, set up in on the insulating substrate, the insulating layer includes first insulating layer and second insulating layer, the insulating substrate the first insulating layer with the second insulating layer is folded in proper order and is established, be provided with the stock solution chamber in the first insulating layer, be provided with in the second insulating layer with the film forming cavity of stock solution chamber intercommunication, the film forming cavity with the stock solution chamber orientation deviates from the direction opening of insulating substrate follows the insulating substrate extremely the direction of second insulating layer, the opening size in film forming cavity crescent.

2. The film-forming support according to claim 1, wherein an inner surface of a first insulating layer forms a liquid storage surface of the liquid storage chamber, an inner surface of a second insulating layer forms a film-forming surface of the film-forming chamber, a top surface of the second insulating layer facing away from the insulating base 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.

3. The film-forming stent of claim 2, wherein the film-forming surface is an inclined or arcuate surface.

4. The film forming stand of claim 2, wherein the second insulating layer further comprises a support protrusion provided to protrude from the film forming surface.

5. The film-forming support according to claim 4, wherein the number of the supporting projections is plural, and the plural supporting projections are arranged in order in a direction from the liquid storage surface to the top surface.

6. The film formation support according to claim 5, wherein each of the plurality of support protrusions includes a support edge and a rising edge, the rising edge extends in a direction from the insulating base to the second insulating layer, the support edges extend along a horizontal plane of the insulating base, and a distance from the support edge to the insulating base of each of the support protrusions arranged in sequence increases gradually in a direction from the liquid storage surface to the top surface.

7. The film forming support according to claim 5, wherein each of the plurality of support protrusions includes a falling edge and a rising edge, the falling edge being provided on a side of the rising edge away from an axis of the film forming chamber,

and the distance from the vertex of the rising edge of each supporting bulge to the insulating base body is gradually increased along the direction from the liquid storage surface to the top surface.

8. The film-forming support according to claim 5, wherein the supporting protrusions include a plurality of first protrusions connected in series to the top surface, and second protrusions connecting the first protrusions and the surface of the liquid reservoir; and the distance from the vertex of each first protrusion to the insulating base body is gradually increased along the direction from the liquid storage surface to the top surface.

9. The film-forming stent according to claim 8, wherein the number of the second protrusions is plural, and the plural second protrusions are connected in series; the vertex of each second protrusion is on the same plane along the direction from the axis of the film forming cavity to the edge of the film forming cavity.

10. The film-forming stent according to claim 4, further comprising a plurality of microprotrusions provided on at least one of the film-forming surface, the reservoir surface and the support protrusions.

11. The film-forming support according to any one of claims 1 to 10, wherein a liquid storage surface forming the liquid storage chamber is provided in a direction from the insulating base to the second insulating layer.

12. The film forming support according to any one of claims 1 to 10, wherein the liquid storage chamber comprises an upper chamber and a lower chamber, the film forming chamber, the upper chamber and the lower chamber are sequentially communicated, and the size of an opening of the upper chamber is gradually reduced in a direction from the insulating base to the second insulating layer.

13. The film-forming rack of claim 12, wherein the maximum angle between the inner wall surface of the upper chamber and the plane of the insulating substrate is α, wherein 1 ° < α < 75 °.

14. The film-forming support according to any one of claims 1 to 10, wherein a plurality of the liquid storage cavities are arranged in an array on the first insulating layer, a plurality of the film-forming cavities are arranged in an array on the second insulating layer, and adjacent film-forming cavities are communicated with each other through a first channel arranged on the second insulating layer;

15. A method of making a film-forming scaffold, comprising:

etching the photoresist layer through a mask with a preset pattern to form a hollow area communicated with the liquid storage cavity in the photoresist layer, wherein the hollow area is a film forming cavity corresponding to the preset pattern, and preparing the film forming support as claimed in any one of claims 1 to 14.

16. A method of making a film-forming scaffold, comprising:

preparing a scaffold template corresponding to the inverted structure of the film-forming scaffold according to any one of claims 1 to 14;

performing mold-turning transfer printing on the support template by adopting an imprinting technology to prepare the film-forming support according to any one of claims 1 to 14.

17. A biochip, comprising:

a film-forming scaffold according to any one of claims 1 to 14 or prepared according to the method of any one of claims 15 to 16;

the electrode, set up in stock solution chamber, the electrode can with the electrically conductive connection of liquid in the stock solution intracavity, and can with the outside circuit connection of biochip.

18. A method of preparing a molecular membrane, comprising:

use of the film-forming scaffold of any one of claims 1 to 14, or the film-forming scaffold prepared by the method of any one of claims 15 to 16, or the biochip of claim 17 to form a molecular film at the film-forming cavity.

19. A nanopore characterization biomolecule device, comprising a membrane-forming scaffold according to any of claims 1 to 14, a membrane-forming scaffold prepared according to any of claims 15 to 16, or a biochip according to claim 17.

20. Use of the film-forming scaffold of any one of claims 1 to 14, the biochip of claim 17, or the nanopore characterization biomolecule device of claim 19 for biomolecule characterization or for the preparation of a product for biomolecule characterization.