Method for manufacturing a chip device and film forming method

Abstract

The application relates to the chip technical field, in particular to a preparation method of a chip device and a film forming method. The preparation method comprises the following steps: providing a substrate layer, the substrate layer has a surface, and a plurality of micro-pore structures are formed on the surface, the micro-pore structures comprise: second layer micro-pores and first layer micro-pores; adding a first electrolyte into the micro-pore structures, the first electrolyte fills the first layer micro-pores; adding a film solution into the micro-pore structures, the film solution is polymerized and fills the gap between the first electrolyte and the first layer micro-pores; adding a viscous protective solution into the micro-pore structures, the viscous protective solution fills the second layer micro-pores, finally, the viscous protective solution, the film solution and the first electrolyte form a stable sandwich layer structure in the micro-pore structures. The amount of the first electrolyte, the film solution and the viscous protective solution is accurately controlled through centrifugation, so that the consistency of the finally formed film is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chips, in particular to a chip device preparation method and a film forming method. BACKGROUND

[0002] Nanopore proteins need to be stably embedded in a phospholipid or polymer formed film to make the DNA sequence to be detected pass through to achieve the purpose of detecting the DNA sequence. Carrying such a phospholipid or polymer film requires a micropore with a diameter of 100 microns to 200 microns, and a technology for forming a film on the micropore.

[0003] CN113070113A discloses a chip structure, a film forming method, a nanopore sequencing device and an application. The chip structure comprises a substrate layer and an oil discharge channel. The first surface of the substrate layer comprises a first functional film layer structure, and the first functional film layer structure comprises arrayed structure units, and adjacent structure units are in communication with each other; the first end of the oil discharge channel is in communication with the first functional film layer structure, and the oil discharge channel is limited to pass through the non-polar solvent. Adjacent structure units of the first functional film layer structure are in communication with each other, non-polar solvent in the structure unit can exude and be discharged through the oil discharge channel.

[0004] CN104918696B discloses a membrane array comprising amphiphilic molecules is formed using a device comprising a support defining an array of compartments. A volume comprising a polar medium is provided within each compartment and a layer comprising a non-polar medium is provided extending through an aperture having said volume. A polar medium is flowed through said support to displace the non-polar medium and form a layer in contact with said volume, forming a membrane comprising amphiphilic molecules at the interface.

[0005] CN101932933B discloses an element device for forming a layer (11) separating two volumes of aqueous solution, in particular a biological layer lipid membrane (BLM), using an element device comprising a body (2) of non-conductive material in which at least one recess (5) opening into a cavity (7) is formed, said recess comprising an electrode (21). A hydrophobic fluid pre-treatment coating is applied on said body spanning said recess. An aqueous solution having added thereto an amphiphilic molecule is flowed through said body to cover said recess, so that the aqueous solution is introduced from said cavity into said recess, an amphiphilic molecule layer is formed spanning said recess, and the volume of aqueous solution introduced into said recess is separated from the remaining volume of aqueous solution.

[0006] However, the existing film forming structure not only has a high manufacturing difficulty, but also has a complex subsequent film forming and pore embedding operation process, needs to use a plurality of toxic reagents, and has a non-uniform film thickness, some of which is thick and some of which is thin. Moreover, not all films can be embedded in the protein pores, only the amphiphilic molecule layer can be normally embedded in the protein, and the success rate of film forming and pore embedding is not high. Referring to the internationally leading Oxford Nanopore Company, more than 800 films formed and embedded on 2048 micropores can be determined as qualified products, and obviously the effective pore proportion on a chip capable of sequencing is not high. SUMMARY

[0007] In order to solve the above-mentioned technical problems, the application specifically adopts the following technical solutions:

[0008] The application provides a preparation method of a chip device, wherein the chip device comprises a substrate layer, and the preparation method comprises the following steps:

[0009] S1, providing a substrate layer having a surface, and a plurality of micropore structures are formed on the surface, wherein the micropore structures comprise: a second layer of micropores 22 and a first layer of micropores 21 which are recessed inward in a direction away from the surface in sequence, and the diameter of the second layer of micropores 22 is greater than the diameter of the first layer of micropores 21, so that a step is formed adjacent to the first layer of micropores 21, and the step is used for supporting a membrane solution;

[0010] S2, adding a first electrolyte into the micropore structure, and performing first centrifugation at a first rotating speed to remove excess first electrolyte and make the remaining first electrolyte fill the first layer of micropores 21; under the action of centrifugation, the height of the first electrolyte at multiple positions is the same or approximately the same; the first electrolyte is a gel electrolyte;

[0011] S3, adding the membrane solution into the micropore structure, so that the membrane solution fills the first layer of micropores 21 or the membrane solution overflows the first layer of micropores 21, and then performing second centrifugation at a second rotating speed, so that the membrane solution polymerizes and fills the gap between the first electrolyte and the first layer of micropores 21, and partially covers the step; under the action of centrifugation, the content of the membrane solution at multiple positions is the same or approximately the same;

[0012] S4, adding a viscous protective liquid into the micropore structure, and performing third centrifugation at a third rotating speed to make the viscous protective liquid fill the second layer of micropores 22, so that the viscous protective liquid, the membrane solution and the gel electrolyte finally form a stable sandwich layer structure in the micropore structure.

[0013] Preferably, the first electrolyte assumes a convex shape within the first layer of micropores, the highest point of the convex shape being on the same horizontal plane as the first end of the first layer of micropores.

[0014] Preferably, in the sandwiched layer structure, the membrane solution covering the first portion of the step has a first liquid surface, the membrane solution filling the second portion of the gap has a second liquid surface, the first liquid surface and the second liquid surface are in contact with the viscous protective liquid, so that the first liquid surface and the second liquid surface form an included angle a in the range of 98-130°.

[0015] In some embodiments, the included angle a is in the range of 103-113° or 115-125°.

[0016] The viscous protective liquid comprises a water-soluble high molecular polymer and / or a water-soluble non-ionic high molecular compound.

[0017] Specifically, the water-soluble high molecular polymer comprises polyacrylic acid (PAA).

[0018] Specifically, the water-soluble non-ionic high molecular compound comprises polyvinylpyrrolidone (PVP).

[0019] Further, the polyvinylpyrrolidone (PVP) comprises PVP K30 and / or PVP K60.

[0020] In some embodiments, the first layer of micropores 21 has a structural thickness of 60-150 μm and a diameter of 70-120 μm, and the second layer of micropores 22 has a structural thickness of 20-50 μm and a diameter of 90-150 μm.

[0021] Preferably, the first electrolyte has viscosity, and the gel component in the first electrolyte is methacrylated gelatin (GelMA) or methacrylated hyaluronic acid gelatin (HAMA), and the concentration of the gel component is in the range of 5-15%.

[0022] Further, the solute of the membrane solution is phospholipid, artificially synthesized high molecular membrane, or a mixture of phospholipid and high molecular membrane; and the solvent of the membrane solution is a non-polar solvent, which comprises AR20, silicone oil, hexadecane, decane, or a mixture thereof.

[0023] In addition, the preparation method further comprises cutting the base layer and assembling a flow channel structure using a cover layer.

[0024] The application also provides a film forming method, which comprises:

[0025] A chip device is provided, a flow channel structure is formed in the chip device, and the chip device is prepared by using the preparation method.

[0026] At least one cleaning solution is injected into the flow channel structure, so that the cleaning solution enters the second layer of micropores to dilute the viscous protective solution;

[0027] A second electrolyte is injected into the flow channel structure, so that the second electrolyte enters the second layer of micropores and replaces the cleaning solution, and fills the second layer of micropores; wherein the amphiphilic material in the membrane solution is automatically arranged to form an amphiphilic molecular layer under the action of the first electrolyte and the second electrolyte.

[0028] Beneficial effects:

[0029] It is worth noting that the present application proposes a special process based on multi-stage centrifugal addition of film-forming material (i.e. amphiphilic material), which can accurately and quantitatively control the film-forming material and the specific solution (such as viscous protective solution) for protecting the film-forming material through multi-stage centrifugal process, and this multi-stage centrifugal addition method cooperates with the special micropore design to ensure long-term stable storage of the membrane solution.

[0030] Further, the present application proposes a film solution distribution scheme with thick edges and thin middle (i.e. the upper surface of the gel electrolyte is convex, so that the membrane solution fills the lower area on both sides), and the applicant notices that in this special thickness distribution state, the final "amphiphilic molecular layer" that meets the standard can be stably formed in the central area of the circular membrane, and at this time the consistency of the film formed by the membrane solution in the multiple holes is very high.

[0031] Moreover, this method of adding film-forming material based on multi-stage centrifugation unexpectedly improves the film-forming success rate to more than 90%, and the hole embedding success rate can also reach more than 90%. The film-forming device developed by the traditional Oxford Nanopore Company is considered to be qualified when more than 800 micropores are formed and embedded on 2048 micropores, so the existing film-forming and embedding difficulty is very high, and the effective hole ratio of the chip device prepared by the preparation method of the present application is very high.

[0032] In summary, the present application designs a preparation method of a chip device based on a sandwich film-forming structure based on the process of adding film-forming material by multi-stage centrifugation, which not only maintains the stability of the membrane solution form during the chip preparation process and the cleaning process, but also is beneficial to the long-term stable storage of the membrane solution, and also ensures the consistency of the membrane solution in the micropore array, thereby making the film-forming efficiency of the chip device high, the uniformity of the amphiphilic molecular layer formed in the micropore array better, and the consistency higher.

[0033] Specifically, the present application uses a multi-stage centrifugal process to accurately and quantitatively control the amount of the first electrolyte, the membrane solution and the viscous protective solution in each micropore structure, which is conducive to improving the success rate of membrane formation in the micropore array. First, the first electrolyte is in a convex shape in the first layer of micropores, so that the membrane solution can fill the lower areas on both sides, which can better support the membrane solution and is conducive to the formation of the amphiphilic molecule layer; second, the membrane solution after polymerization presents the characteristics of thick edges and thin middle, so that the final standard amphiphilic molecule layer is mainly formed in the central area of the circular membrane, which is conducive to improving the uniformity and consistency of the amphiphilic molecule layer formed in the micropore array; finally, the membrane solution and the first electrolyte are locally sealed by the viscous protective solution, which does not affect the chemical properties and physical form of the membrane solution (i.e. through the cooperation of the specific viscous protective solution and the local sealing scheme, the membrane solution form can maintain high consistency and stability during device transportation and use, thereby facilitating the improvement of the subsequent success rate of membrane formation and the consistency of the membrane). Moreover, the viscous protective solution is less toxic and friendly to production personnel.

[0034] In addition, the sandwich membrane formation structure formed by the first electrolyte, the membrane solution and the viscous protective solution is also conducive to further maintaining the good form of the membrane solution during long-term storage. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. In all the drawings, similar elements or parts are generally identified by similar reference signs. In the drawings, each element or part is not necessarily drawn according to the actual proportion. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without any creative labor.

[0036] Figure 1 A schematic diagram of a chip device in an exemplary embodiment of the present application;

[0037] Figure 2 A schematic diagram of a two-layer micropore structure prepared in an exemplary embodiment of the present application;

[0038] Figure 3 A schematic diagram of a non-solidified gel electrolyte on a micropore structure in an exemplary embodiment of the present application;

[0039] Figure 4 A schematic diagram of the heated gel electrolyte flowing into the micropore structure in an exemplary embodiment of the present application;

[0040] Figure 5A schematic diagram of the gel electrolyte in the first layer of micropores in an exemplary embodiment of the present application;

[0041] Figure 6 A schematic diagram of the gel electrolyte solidified in the micropores and the polymerized membrane solution in an exemplary embodiment of the present application;

[0042] Figure 7a A schematic diagram of the sandwich structure formed by the adhesive protective liquid, the membrane solution and the gel electrolyte in an exemplary embodiment of the present application; Figure 7b A schematic diagram of the included angle a formed by the membrane solution and the adhesive protective liquid in an exemplary embodiment of the present application; Figure 7c A schematic diagram of another double-layer microporous structure in an exemplary embodiment of the present application;

[0043] Figure 8 An electrical characterization diagram of the film formed on the chip device in an exemplary embodiment of the present application.

[0044] The label identification summary: 1, the base layer; 2, the microporous structure, 21, the first layer of micropores, 22, the second layer of micropores; 3, the film forming unit; 4, the first electrolyte; 5, the membrane solution; 6, the adhesive protective liquid; 7, the cover layer; 8, the flow channel structure, 81, the first opening, 82, the second opening. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0046] Herein, the suffix such as "module", "component" or "unit" used to indicate an element is only for the convenience of the description of the present application, and has no specific meaning by itself. Therefore, "module", "component" or "unit" can be used mixedly.

[0047] Herein, the terms "upper", "lower", "inner", "outer", "front", "back", "one end", "the other end" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the indicated device or element must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second" are only for the purpose of description, and cannot be understood as indicating or implying relative importance.

[0048] In this document, unless otherwise indicated and / or unless the context clearly dictates otherwise, the terms "mounting", "provided with", "connected" and the like are to be interpreted broadly, for example, "connected" can be fixed connection, can be detachable connection, or integral connection; can be mechanical connection, can be direct connection, can be indirect connection through intermediate medium, can be internal connection of two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances. In this document, "and / or" includes any and all combinations of one or more listed items.

[0049] In this document, "and / or" includes any and all combinations of one or more listed items.

[0050] In this document, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0051] In this document, the term "about" is typically + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, even more typically + / - 0.5% of the stated value.

[0052] In this document, the term "about" is typically + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, even more typically + / - 0.5% of the stated value.

[0053] In this document, certain embodiments can be disclosed in a format that is a range of values. It is to be understood that such a "range of values" description is merely a shorthand for describing a set of individual values, and is simply a shorthand for describing a set of individual values. For example, a description of a range of values 1-6 should be considered to have specifically disclosed the sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0054] Example 1

[0055] The present application provides a chip device preparation method, and the specific preparation method is as follows:

[0056] S1, providing a substrate layer, the substrate layer has a surface, and a plurality of micro-hole structures are formed on the surface, and the micro-hole structures correspondingly comprise: a second layer of micro-holes 22 and a first layer of micro-holes 21 are sequentially formed by being recessed inward in a direction away from the surface, and the diameter of the second layer of micro-holes 22 is greater than the diameter of the first layer of micro-holes 21, so that the second layer of micro-holes 22 is formed with a step adjacent to the first layer of micro-holes 21, and the step is used to support the membrane solution;

[0057] S2, adding a first electrolyte into the micro-hole structure, and performing a first centrifugation at a first rotating speed to remove excess first electrolyte and make the remaining first electrolyte fill the first layer of micro-holes 21; under the action of centrifugation, the height of the first electrolyte at multiple places is the same or approximately the same; the first electrolyte is a gel electrolyte;

[0058] S3, adding the membrane solution into the micro-hole structure, so that the membrane solution fills the first layer of micro-holes 21 or the membrane solution overflows the first layer of micro-holes 21, and then performing a second centrifugation at a second rotating speed, so that the membrane solution is polymerized and filled in the gap between the first electrolyte and the first layer of micro-holes 21, and partially covers the step; under the action of centrifugation, the content of the membrane solution at multiple places is the same or approximately the same;

[0059] S4, adding a viscous protective liquid into the micro-hole structure, and performing a third centrifugation at a third rotating speed to make the viscous protective liquid fill the second layer of micro-holes 22, and finally the viscous protective liquid, the membrane solution and the gel electrolyte form a stable sandwich layer structure in the micro-hole structure.

[0060] The sandwich layer structure can not only maintain the good morphology of the membrane solution in the micro-holes during the chip preparation process and the cleaning process, but also maintain the consistency of the membrane solution in the micro-hole array.

[0061] Preferably, the present application forms a sandwich layer structure on the substrate layer by using a multi-stage centrifugation process, which on the one hand utilizes the centrifugation process and the special micro-hole structure (which is designed to be large at the top and small at the bottom) to precisely arrange the content and morphology of each layer of the sandwich layer, and on the other hand uses the viscous protective liquid to seal the membrane solution in the local area (i.e. in the micro-hole structure), which can not only improve the stability of the sandwich layer structure, but also simplify the subsequent processing difficulty (such as the cooperation between the substrate layer and the cover layer during processing will not affect the stability of the sandwich layer structure).

[0062] Preferably, the first electrolyte in the first layer of micro-holes presents a convex shape, and the highest part of the convex shape is at the same level as the first end of the first layer of micro-holes.

[0063] Preferably, in the sandwich structure, the first portion of the membrane solution covering the step has a first liquid surface, the second portion of the membrane solution filling the gap has a second liquid surface, and the first liquid surface and the second liquid surface are in contact with the viscous protective liquid such that the first liquid surface and the second liquid surface form an angle α.

[0064] In some embodiments, the included angle α ranges from approximately 115-125°. The applicant notes that when an inverted trapezoidal microporous structure design is used, the support provided by the second layer of micropores is particularly beneficial for the two liquid surfaces of the membrane solution to form an included angle of approximately 115-125° (preferably 120°).

[0065] Among them, the first electrolyte, membrane solution, and viscous protective liquid can form a stable state with lower energy within this specific angle range (in other words, under the dual compression of the first electrolyte and the viscous protective liquid, the cross-section of the liquid surface in contact with the membrane solution and the viscous protective liquid forms a geometric shape similar to half of a regular hexagon, at which point the forces between the liquid surfaces are more balanced, and the system energy is also lower).

[0066] Of course, in other embodiments, a local geometric shape resembling a pentagon can also be formed between the first liquid surface and the second liquid surface (such as several liquid surfaces in contact with the viscous protective liquid, such as the first liquid surface (e.g.)). Figure 7b The liquid surface shown on the left), the second liquid surface, the first liquid surface (as shown on the left) Figure 7b The liquid surface shown on the right approximates the three adjacent sides of a pentagon, and the included angle α can be approximately 103-113°. In this case, the membrane solution can still maintain a relatively stable state.

[0067] Furthermore, in some embodiments, the included angle α (e.g.) Figure 7b The value (as shown) can also be approximately 98-130° to enable the membrane solution to form a stable form with lower energy.

[0068] Furthermore, the viscous protective liquid comprises water-soluble polymers and / or water-soluble nonionic polymers.

[0069] Specifically, the water-soluble polymer includes polyacrylic acid (PAA).

[0070] Specifically, the water-soluble nonionic polymeric compound includes polyvinylpyrrolidone (PVP).

[0071] Furthermore, the polyvinylpyrrolidone (PVP) includes PVP K30 and / or PVP K60. The electrical characterization diagram of the chip device after film deposition based on PVP K30 is shown below. Figure 8 As shown.

[0072] In some embodiments, the first layer of micro-holes 21 has a structural thickness of 60-150 μm and a diameter of 70-120 μm; and the second layer of micro-holes 22 has a structural thickness of 20-50 μm and a diameter of 90-150 μm.

[0073] Preferably, the first electrolyte has viscosity, and the gel component in the first electrolyte is methacrylated gelatin (GelMA) or methacrylated hyaluronic acid gelatin (HAMA), and the concentration of the gel component ranges from 5% to 15%.

[0074] Further, the solute of the membrane solution is phospholipid, artificially synthesized polymer membrane, or a mixture of phospholipid and polymer membrane; and the solvent of the membrane solution is a non-polar solvent, which includes AR20, silicone oil, hexadecane, decane, or a mixture thereof.

[0075] In addition, the preparation method further includes cutting the substrate layer and assembling the flow channel structure by using the cover layer.

[0076] The present application also provides a film forming method, which includes:

[0077] The present application also provides a chip device, which is formed with a flow channel structure and is prepared by using the above preparation method.

[0078] The flow channel structure is injected with at least one cleaning liquid (such as water) so that the cleaning liquid enters the second layer of micro-holes to dilute the viscous protective liquid.

[0079] The flow channel structure is injected with a second electrolyte so that the second electrolyte enters the second layer of micro-holes to replace the cleaning liquid and fill the second layer of micro-holes; and the amphiphilic material (such as amphiphilic molecules) in the membrane solution is automatically arranged to form an amphiphilic molecule layer under the action of the first electrolyte and the second electrolyte.

[0080] In some embodiments, the cleaning liquid can also be an electrolyte.

[0081] The following embodiments provide a specific example of a preparation method of a chip device.

[0082] (1) A micro-hole structure 2 is prepared on a silicon wafer by using SU-8 photoresist and a method of spinning glue (as shown in FIG. 1), which includes a second layer of micro-holes 22 and a first layer of micro-holes 21 formed in sequence in a direction of inwardly recessing away from the surface of the silicon wafer. Figure 2

[0083] ​Preferably, the first layer of micro-holes 21 has a thickness ranging from 60 to 150 μm and a diameter ranging from 70 to 120 μm; the second layer of micro-holes 22 has a thickness ranging from 20 to 50 μm and a diameter ranging from 90 to 150 μm.

[0084] The diameter of the second layer of micro-holes 22 is larger than that of the first layer of micro-holes 21, so that the second layer of micro-holes 22 forms a step adjacent to the first layer of micro-holes 21, which helps to stably support the subsequent film solution.

[0085] Referring to Figure 7b As shown, the double-layer micro-hole structure according to the present application can promote the film solution to be stored in a stable form with low energy. Specifically, the film solution covering the first part of the step forms a first liquid surface, the film solution filling the gap forms a second liquid surface, the first liquid surface and the second liquid surface are in contact with the viscous protective liquid, and the first liquid surface and the second liquid surface form an alpha angle ranging from 98 to 130°, so that the film solution can be polymerized to form a stable form with low energy.

[0086] In another exemplary embodiment of the present application, referring to Figure 7c As shown, the cross section of the first layer of micro-holes is square, and the cross section of the second layer of micro-holes is inverted trapezoidal. This structure, which is large at the top and small at the bottom, can form an alpha angle of about 115-125°, and can also enhance the support of the film solution, ensuring that the film solution remains in a stable state with low energy during long-time transmission.

[0087] In some embodiments, a dry film of SUEX or AUEX photoresist can be directly laminated on a silicon wafer (i.e., a substrate layer) to prepare a two-layer micro-hole structure.

[0088] In some other embodiments, a nano-imprint method can be used to heat a special imprinting glue to about 1200°C to imprint the desired two-layer micro-hole structure, and then the structure can be demolded after the temperature decreases.

[0089] (2) The gel electrolyte 4 (i.e., the first electrolyte) is added dropwise into the micro-hole structure 2. The initially uncured gel electrolyte has a relatively large viscosity and cannot enter the micro-hole structure (as shown in Figure 3 After the silicon wafer is heated to about 90°C on a hot plate, the viscosity of the uncured gel electrolyte 4 decreases and flows into the micro-hole structure, as shown in Figure 4

[0090] ​Then the silicon wafer is placed on a spin coater, using a rotation speed of 500-1000 rpm, and the gel electrolyte is spun for 2-10 minutes to remove the excess un-solidified gel electrolyte from the surface and inside of the microporous structure under the action of centrifugal force, so as to keep the height of the gel electrolyte in the microporous array consistent, and the remaining gel electrolyte fills the first layer of micropores 21.

[0091] In other words, by adjusting the rotation speed, the height of the gel electrolyte can be adjusted. The higher the rotation speed, the more the gel is spun away by the centrifugal force, and the lower the liquid level.

[0092] After the spinning is completed, the silicon wafer is left to stand for 5 minutes and naturally cooled to room temperature. Due to the hydrophobicity of the material of the inner wall of the micropores, the gel electrolyte in the first layer of micropores assumes a convex shape (as shown in Figure 5 ), and the highest point of the convexity is at the same level as the first end of the first layer of micropores.

[0093] An ultraviolet curing light source with a wavelength of 365 nm is used to cure the silicon wafer for 30 seconds at a height of 5 cm. The cured gel electrolyte can better support the film solution, and the height of the gel electrolyte in each micropore is consistent.

[0094] Preferably, the gel component in the gel electrolyte is methacrylated gelatin (GelMA) or methacrylated hyaluronic acid gelatin (HAMA), and the concentration range is 5-15%.

[0095] (3) The film solution 5 is prepared, and the film-forming material of the film solution 5 includes phospholipids, artificially synthesized high molecular films, or a mixture of phospholipids and high molecular films.

[0096] The film-forming material can be dissolved in AR20, silicone oil, hexadecane, decane, or a mixture thereof.

[0097] (4) The film solution 5 is added to the microporous structure 2. The film solution will first fill the lower part of the first layer of micropores 21 to facilitate the formation of the film, and finally fill the first layer of micropores 21 or overflow the first layer of micropores 21. The silicon wafer is placed on a spin coater and spun at a rotation speed of 100-300 rpm to remove the excess film solution under the action of centrifugal force. Under the action of centrifugal force, the content of the film solution in the microporous array is kept the same or approximately the same. At this time, there is a small amount of film solution 5 on the side wall of the micropore.

[0098] Further, the substrate layer to which the film solution is added can also be subjected to cold storage treatment at a temperature of 2-8°C. For example, the silicon wafer can be stored in a refrigerator at 4°C for 30 minutes. The film solution will slowly polymerize and spread on the gel electrolyte 4 in the micropore (as shown in Figure 6 ).

[0099] (5) Add viscous protective liquid 6 into the micro-porous structure, remove the excess viscous protective liquid by centrifugation, so that the viscous protective liquid fills the second layer of micro-pores, and finally the viscous protective liquid, the membrane solution and the first electrolyte form a stable sandwich layer structure (i.e. a sandwich film forming structure, as shown in Figure 7a

[0100] Further, the viscous protective liquid comprises polyacrylic acid (PAA) and / or polyvinylpyrrolidone (PVP).

[0101] Further, the viscous protective liquid is preferably PVP K30. The PVP K30 solution is dropped onto a silicon wafer, and a glue spinner is used to spin the glue at a speed of 300-500 rpm for 15 minutes to remove the excess PVP K30, and the effect is as shown in Figure 7a

[0102] Preferably, when PVP K30 is selected as the viscous protective liquid material, 30% by mass of PVP K30 and 70% by mass of pure water can be mixed during use, and the beaker used for mixing is placed on a shaker at a speed of 100 rpm for 1 hour at room temperature until it is completely dissolved. Alternatively, a PVP K30 solution with a mass fraction of 30% can be purchased directly.

[0103] Of course, in other embodiments, the user can also select solutions with different masses according to different application environments, different film forming materials and different film forming standards.

[0104] (6) Cut the silicon wafer, use a waterproof gasket, a clamp, and assemble a flow channel structure by screws, and the device formed is as shown in Figure 1

[0105] After the device is assembled, the cleaning liquid and the second electrolyte can be injected into the flow channel to form a film.

[0106] In some embodiments, the cleaning liquid is pure water, and 300 μL of pure water is injected into the flow channel, at which time the PVP K30 is diluted by the pure water. Every 5 minutes, 1000 μL of pure water is used to slowly flush the flow channel, and this is repeated three times. The fourth time is to pass 300 μL of electrolyte containing protein, at which time the PVP K30 aqueous solution is completely washed clean by the pure water, and the membrane solution just forms an amphiphilic molecule film (or an amphiphilic molecule layer, also simply referred to as "film") between the gel electrolyte and the electrolyte containing protein, and the protein can normally embed in the amphiphilic molecule film to form channels on the film.

[0107] ​​​DNA passes through the protein channel under the action of the electric field, and according to the number of blocked channels, the current between the two ends of the membrane changes under the applied voltage, and according to the change of the current, it is known which sequence of DNA passes through the channel, thereby realizing sequencing.

[0108] The film forming effect can be detected by electrical characterization of the membrane capacitance. The thickness of the film formed in each micropore can be determined by measuring the capacitance of the film, and the thickness of the formed amphiphilic molecular layer is different, and the electrical characterization is also different. The micropore array of the present application has 1024 micropores, which are divided into 4 areas, AREA1 to AREA4, and the number of membrane capacitances that can be measured at a time is 256. The present application selects each AREA to measure all 1024 micropores in steps. Figure 8 Each rectangular block in the figure represents a membrane capacitance corresponding to a micropore structure, and the numerical value in the rectangular block represents the capacitance value of the membrane capacitance.

[0109] And the display color of each rectangular electrical characterization in the instrument has a certain relationship with the size of the membrane capacitance value, and the numerical value of the capacitance value can represent the state of different micropores, such as whether the film is formed, and the thickness of the formed film layer. Specifically, the capacitance value less than 40 pF is displayed as green, indicating that the formed amphiphilic molecular film has too large thickness; the capacitance value in the range of 40-70 pF is displayed as light gray, indicating that the formed amphiphilic molecular film has appropriate thickness; and the capacitance value greater than 70 pF is displayed as red, indicating that the formed amphiphilic molecular film has too small thickness.

[0110] Selecting different AREA on the right, the 256 micropores of the AREA will be displayed in the left for easy reading of the specific membrane capacitance value. As shown in Figure 8 The film forming result shows that the membrane capacitances of the 1024 micropores are in the range of 40-70 pF, indicating that the formed amphiphilic molecular film has appropriate thickness, which is conducive to subsequent conventional hole embedding.

[0111] The results show that the use of the preparation method of the present application greatly improves the film forming success rate on the multi-channel micropore. Using membrane capacitance detection, all micropores are fully formed, the film consistency is good, the hole embedding rate is significantly improved to more than 90%, and the effective hole ratio is greatly improved.

[0112] Further, in order to verify the film forming efficiency of the present application, 20 workers collected comprehensive evaluation of different adhesive protective solutions, the evaluation content including: protection effect, operation efficiency, operation difficulty and the most important membrane capacitance value, which can judge the thickness of the film. The results are shown in Table 1, wherein the protection effect reflects the stability of the film solution form in the assembly and cleaning stages of the chip, the operation efficiency reflects the stage of passing different reagents, and the operation difficulty reflects the protection of the operator and the influence on the environment after the operation of the reagent.

[0113] Table 1 Comprehensive evaluation results of different viscous protective liquids by workers

[0114]

[0115] As can be seen from Table 1, the use of PAA and PVP protective liquid materials significantly improves the final film embedding hole rate; it is worth noting that in the film forming device promoted by Oxford Nanopore, more than 800 micro-holes are formed on 2048 micro-holes, which is considered to be qualified, that is, the embedding hole success rate reaches 39%, which can be considered to be qualified, so it can be seen that the existing film forming process is very difficult, and the embedding hole success rate is very low. On the contrary, the multi-stage centrifugal process, special micro-hole design and special protective liquid used in the present application unexpectedly directly improve the embedding hole success rate to about 88%.

[0116] Further, PVP directly improves the embedding hole success rate to more than 91%, and the operation efficiency and operation difficulty comprehensively meet the application requirements. In particular, PVP K30 can reach 100% embedding hole success rate in a better state, and has low operation difficulty and high operation efficiency.

[0117] The present application innovatively uses a glue throwing process (i.e. a multi-stage centrifugal process) to add film forming materials. In view of the fact that the chips used in the current nanopore sequencing field are first processed by micro-nano processing, and then complex micro-fluidic operations and various reagents are used to form a film and embed a hole, the present application creatively fuses the film forming material in the chip manufacturing process by micro-nano technology, and then only needs to pass pure water into the flow channel to form a film. The film forming success rate is more than 90% (even more than 95%, for example, 100%), and the embedding hole success rate can reach 90% (even more than 95%, for example, 100%).

[0118] Example 2

[0119] Based on the preparation method of Example 1, the present application provides a chip device, which comprises:

[0120] a substrate layer 1, at least one micro-hole structure 2 is arranged on the substrate layer 1;

[0121] The micro-hole structure 2 comprises a first layer of micro-holes 21 and a second layer of micro-holes 22, wherein the second layer of micro-holes 22 is located on the upper layer of the first layer of micro-holes 21;

[0122] For example, the substrate layer has a surface, and a plurality of micro-hole structures are formed on the surface, and the micro-hole structures include: a second layer of micro-holes 22 and a first layer of micro-holes 21 which are recessed inwardly in a direction away from the surface and sequentially formed, and the diameter of the second layer of micro-holes 22 is greater than the diameter of the first layer of micro-holes 21, so that the second layer of micro-holes 22 is formed with a step adjacent to the first layer of micro-holes 21, and the step is used to support the film solution.

[0123] For example, in some embodiments, the diameter of the second end of the second layer of micro-holes is greater than the diameter of the first layer of micro-holes, and at this time, the plane formed at the connection of the second layer of micro-holes and the first layer of micro-holes is the step. Specifically, the cross section (i.e. the plane perpendicular to the surface) of the second layer of micro-holes can be rectangular or approximately rectangular.

[0124] For another example, in some embodiments, the diameter of the second layer of micro-holes gradually decreases in the direction from the first end to the second end, and at this time, the second end of the second layer of micro-holes forms the step (i.e. the slope) to support the film solution.

[0125] In other words, in this embodiment, the cross section of the second layer of micro-holes is in the shape of an inverted trapezoid. The design of the inverted trapezoid is conducive to the formation of an included angle α of about 115-125° (preferably 120°) between the two liquid surfaces of the film solution at the step (as shown in Figure 7b At this time, the first electrolyte, the film solution and the viscous protective liquid can form a stable form with low energy.

[0126] In other embodiments, a local geometric figure similar to a pentagon can be formed between the two sides (such as the two first liquid surfaces) of the film solution in contact with the viscous protective liquid and the liquid surface (such as the second liquid surface) formed by the film solution filling the first layer of micro-holes, and the included angle α formed thereby is about 103-113°.

[0127] Further, the method further includes: forming a film unit 3, and the film unit 3 includes: a first electrolyte 4, a film solution 5 and a viscous protective liquid 6, wherein,

[0128] The first electrolyte 4 is in the first layer of micro-holes 21, and after solidification, it presents a convex form to better support the film solution; the film solution 5 is polymerized and spread on the upper surface of the first electrolyte 4; the viscous protective liquid 6 fills the second layer of micro-holes 22, and the film solution 5 is between the viscous protective liquid 6 and the first electrolyte 4, and the viscous protective liquid 6, the film solution 5 and the first electrolyte 4 form a stable sandwich structure to maintain the form of the film solution 5 in the micro-holes and the form consistency in the micro-hole array;

[0129] The chip device further comprises a cover layer 7, and the cover layer 7 and the base layer 1 assemble a flow channel structure 8, the flow channel structure 8 is respectively provided with a first opening 81 and a second opening 82.

[0130] Through the above description of the embodiments, those skilled in the art can clearly understand that the above-mentioned example method can be realized by means of software and the necessary general hardware platform, of course, it can also be realized by hardware, but in many cases, the former is a better embodiment. Based on such understanding, the technical solutions of the present application can be embodied in the form of a software product, which is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk) and includes a plurality of instructions for causing a computer terminal (which can be a mobile phone, computer, server, or network device) to execute the method described in each embodiment of the present application.

[0131] It should be noted that in this document, the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusion, so that processes, methods, articles or devices including a series of elements not only include those elements, but also include other elements not explicitly listed, or further include elements inherent to such processes, methods, articles or devices. Without more limitations, the element defined by the statement "including a" does not exclude the presence of other identical elements in the process, method, article or device including the element.

[0132] The embodiments of the present application are described above in combination with the drawings, but the present application is not limited to the above-mentioned specific embodiments, the above-mentioned specific embodiments are only illustrative, not restrictive, and those skilled in the art can make many forms under the inspiration of the present application without departing from the purpose of the present application and the scope protected by the claims, which all belong to the protection of the present application.

Claims

1. A method for fabricating a chip device, characterized in that, The chip device includes a substrate layer, and correspondingly, the fabrication method includes: S1, a substrate layer is provided, the substrate layer having a surface and a plurality of microporous structures formed on the surface, the corresponding microporous structures including: a second layer of micropores (22) and a first layer of micropores (21) formed sequentially by being recessed inward in a direction away from the surface, and the diameter of the second layer of micropores (22) is larger than the diameter of the first layer of micropores (21), such that a step is formed in the second layer of micropores (22) adjacent to the first layer of micropores (21), the step being used to support the membrane solution; S2, add the first electrolyte into the microporous structure, and centrifuge for the first time at the first speed to remove excess first electrolyte and make the remaining first electrolyte fill the first layer of micropores (21); under centrifugation, the height of the first electrolyte is the same in multiple places; the first electrolyte is a gel electrolyte; S3, the membrane solution is added into the microporous structure so that the membrane solution fills the first layer of micropores (21) or overflows the first layer of micropores (21). A second centrifugation is performed using a second rotation speed. Subsequently, the membrane solution polymerizes and fills the gap between the first electrolyte and the first layer of micropores (21), and partially covers the step. Under centrifugation, the content of the membrane solution is the same in multiple places. S4, a viscous protective liquid is added into the microporous structure, and a third centrifugation is performed at a third rotation speed so that the viscous protective liquid fills the second layer of micropores (22). Finally, the viscous protective liquid, the membrane solution, and the first electrolyte form a stable sandwich structure in the microporous structure. The first electrolyte presents a convex shape in the first layer of micropores, and the highest point of the convexity is on the same horizontal plane as the first end of the first layer of micropores. Alternatively, the structural thickness of the first layer of micropores (21) is 60-150 μm and the diameter is 70-120 μm. The structural thickness of the second layer of micropores (22) is 20-50 μm and the diameter is 90-150 μm. In the sandwich structure, the first part of the membrane solution covering the step has a first liquid surface, and the second part of the membrane solution filling the gap has a second liquid surface. The first liquid surface and the second liquid surface are in contact with the viscous protective liquid so that the first liquid surface and the second liquid surface form an angle α with an angle range of 98°-130°.

2. The preparation method according to claim 1, characterized in that, The included angle α is 103°-113° or 115°-125°.

3. The preparation method according to claim 1, characterized in that, The viscous protective liquid includes water-soluble polymers and / or water-soluble nonionic polymers.

4. The preparation method according to claim 3, characterized in that, The water-soluble polymer includes polyacrylic acid; and / or, the water-soluble nonionic polymer includes polyvinylpyrrolidone.

5. The preparation method according to claim 4, characterized in that, The polyvinylpyrrolidone includes PVP K30 and / or PVP K60.

6. The preparation method according to claim 1, characterized in that, The first electrolyte is viscous, and the gel component in the first electrolyte is methacrylated gelatin or methacrylated hyaluronic acid gelatin, with a concentration range of 5-15%.

7. The preparation method according to claim 1, characterized in that, The solute of the membrane solution includes phospholipids, or synthetic polymer membranes, or a mixture of phospholipids and polymer membranes; the solvent of the membrane solution is a nonpolar solvent, including AR20, silicone oil, hexadecane, or decadecane; or, the preparation method further includes cutting the substrate layer and assembling the flow channel structure using the cover plate layer.

8. A film-forming method, characterized in that, The film-forming method includes: A chip device is provided, wherein a flow channel structure is formed therein, and the chip device is prepared by any one of the preparation methods described in claims 1-7; At least one cleaning solution is injected into the flow channel structure so that the cleaning solution enters the second layer of micropores to dilute the viscous protective solution; A second electrolyte is injected into the flow channel structure so that the second electrolyte enters the second layer of micropores and replaces the cleaning solution, and fills the second layer of micropores; wherein, the amphiphilic material in the membrane solution automatically arranges to form an amphiphilic molecular layer under the action of the first electrolyte and the second electrolyte.

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