Preparation method and film forming method of chip device

Abstract

The invention relates to the technical field of chips, in particular to a preparation method and a film forming method of a chip device. The preparation method comprises the steps that a substrate layer is provided, the substrate layer is provided with a surface, a plurality of micropore structures are formed on the surface, and each micropore structure comprises a second layer of micropores and a first layer of micropores; adding a first electrolyte into the microporous structure, wherein the first layer of micropores are filled with the first electrolyte; adding a membrane solution into the microporous structure, wherein the membrane solution is polymerized and fills gaps between the first electrolyte and the first layer of micropores; and a viscous protection solution is added into the microporous structure, the second layer of micropores are filled with the viscous protection solution, and finally the viscous protection solution, the membrane solution and the first electrolyte form a stable sandwich layer structure in the microporous structure. And the amounts of the first electrolyte, the membrane solution and the viscous protection solution are accurately controlled through centrifugation, so that the consistency of the finally formed membrane is ensured.

Description

Technical Field

[0001] The present invention relates to the field of chip technology, and in particular to a chip device preparation method and a film forming method. Background Art

[0002] Nanopore proteins need to be stably embedded in a phospholipid or polymer film to allow the DNA sequence to pass through. To carry this phospholipid or polymer film, a micropore with a diameter of 100 to 200 microns and the technology to form a film on the micropore are required.

[0003] CN113070113A discloses a chip structure, a film forming method, a nanopore sequencing device and an application, wherein the chip structure comprises: a substrate layer and an oil drainage channel. The first surface of the substrate layer comprises a first functional film layer structure, wherein the first functional film layer structure comprises structural units distributed in an array, and adjacent structural units are connected to each other; the first end of the oil drainage channel is connected to the first functional film layer structure, and the oil drainage channel limits the passage of non-polar solvents. Adjacent structural units of the first functional film layer structure are connected to each other, and the non-polar solvents in the structural units can seep out and be discharged through the oil drainage channel.

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

[0005] CN101932933B discloses an element device, in order to form a layer (11) separating two volumes of aqueous solution, in particular a biolayer lipid membrane (BLM), an element device comprising a defined chamber (7) is used, the element comprising a body (2) of a non-conductive material in which at least one groove (5) leading to the chamber is formed, the groove comprising an electrode (21). A hydrophobic fluid pretreatment coating is applied to the body across the groove. An aqueous solution to which amphiphilic molecules are added flows through the body and covers the groove, so that the aqueous solution is introduced from the chamber into the groove, an amphiphilic molecule layer is formed across the groove, and the volume of the aqueous solution introduced into the groove is separated from the rest of the volume of the aqueous solution.

[0006] However, the existing film-forming structure not only has a difficult manufacturing process, but also has a complicated subsequent film-forming pore-embedded operation process, requiring the use of a variety of toxic reagents. In addition, the thickness of the formed membrane is uneven, some are thicker, some are thinner, and not all membranes can be embedded in protein pores. Only amphiphilic molecular layers can normally embed proteins, and the success rate of film-forming pores is not high. Referring to the internationally technologically advanced Oxford Nanopore Company, a qualified product can be identified if there are more than 800 film-forming pores on 2048 micropores. Obviously, the proportion of effective pores that can be sequenced on a chip is not high. Summary of the invention

[0007] In order to solve the above-mentioned technical problems, the present invention specifically adopts the following technical solutions: The present invention provides a method for preparing a chip device, wherein the chip device comprises: a base layer, and correspondingly, the preparation method comprises: S1, providing a substrate layer, the substrate layer having a surface, and a plurality of microporous structures formed on the surface, and correspondingly the microporous structures include: being concave inwardly in a direction away from the surface to sequentially form a second layer of micropores 22 and a first layer of micropores 21, and the diameter of the second layer of micropores 22 is larger than the diameter of the first layer of micropores 21, so that a step is formed at the second layer of micropores 22 adjacent to the first layer of micropores 21, and the step is used to support a membrane solution; S2, adding a first electrolyte into the microporous structure, and performing a first centrifugation at a first speed to remove excess of the first electrolyte, and allowing the remaining first electrolyte to fill the first layer of micropores 21; under the action of centrifugation, the heights of the first electrolyte at multiple locations are the same or approximately the same; the first electrolyte is a gel electrolyte; S3, adding the membrane solution into the microporous structure so that the membrane solution fills the first layer of micropores 21 or the membrane solution overflows the first layer of micropores 21, performing a second centrifugation at a second speed, and then 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 contents of the membrane solution at multiple locations are the same or approximately the same; S4, adding a viscous protective liquid into the microporous structure, and performing a third centrifugation at a third speed so that the viscous protective liquid fills the second layer of micropores 22, and finally the viscous protective liquid, the membrane solution and the gel electrolyte form a stable sandwich structure in the microporous structure.

[0008] Preferably, the first electrolyte is in the form of a protrusion in the first layer of micropores, and the highest point of the protrusion is on the same horizontal plane as the first end of the first layer of micropores.

[0009] Preferably, in the sandwich layer structure, the first part of the membrane solution covering the step has a first liquid level, and the second part of the membrane solution filling the gap has a second liquid level, and the first liquid level and the second liquid level are in contact with the viscous protective liquid so that the first liquid level and the second liquid level form an angle α in the range of 98-130°.

[0010] In some embodiments, the angle α is 103-113° or 115-125°.

[0011] The viscous protective liquid includes a water-soluble high molecular polymer and / or a water-soluble non-ionic high molecular compound.

[0012] Specifically, the water-soluble high molecular polymer includes polyacrylic acid (PAA).

[0013] Specifically, the water-soluble nonionic polymer compound includes polyvinyl pyrrolidone (PVP).

[0014] Furthermore, the polyvinyl pyrrolidone (PVP) includes PVP K30 and / or PVP K60.

[0015] In some embodiments, the first layer of micropores 21 has a structural thickness of 60-150 μm and a diameter of 70-120 μm; the second layer of micropores 22 has a structural thickness of 20-50 μm and a diameter of 90-150 μm.

[0016] Preferably, the first electrolyte is viscous, and the gel component in the first electrolyte is methacryloyl gelatin (GelMA) or methacryloyl hyaluronic acid gelatin (HAMA), and the concentration range of the gel component is 5-15%.

[0017] Furthermore, the solute of the membrane solution is phospholipid, artificial synthetic polymer membrane or a mixture of phospholipid and polymer membrane; the solvent of the membrane solution is a non-polar solvent, and the non-polar solvent includes AR20, silicone oil, hexadecane, decane or a mixture thereof.

[0018] In addition, the preparation method further comprises cutting the base layer and assembling a flow channel structure using a cover layer.

[0019] The present invention also provides a film forming method, which comprises: A chip device is provided, wherein a flow channel structure is formed in the chip device, and the chip device is prepared by the preparation method as described above; Injecting a cleaning liquid into the flow channel structure at least once, so that the cleaning liquid enters the second layer of micropores to dilute the viscous protective liquid; 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.

[0020] Beneficial effects: It is worth mentioning that the present invention proposes a special process based on multi-stage centrifugal addition of membrane-forming materials (i.e., amphiphilic materials). The multi-stage centrifugal process can accurately and quantitatively control the membrane-forming materials and the specific solutions used to protect the membrane-forming materials (such as viscous protective solutions). In addition, this multi-stage centrifugal addition method combined with a special micropore design can ensure long-term stable storage of the membrane solution.

[0021] Furthermore, the present invention proposes a membrane solution distribution scheme that is thick at the edges and thin in the middle (i.e., the upper surface of the gel electrolyte is convex, so that the membrane solution fills the lower areas on both sides thereof). The applicant noticed that under this special thickness distribution state, the "amphiphilic molecular layer" that finally meets the standards can be stably formed in the central area of ​​the circular membrane, and at this time, the consistency of the membrane formed by the membrane solution in multiple holes is very high.

[0022] Moreover, this method of adding film-forming materials based on multi-stage centrifugation unexpectedly increases the success rate of film formation to more than 90%, and the success rate of hole embedding can also reach more than 90%. The film-forming device launched by the traditional Oxford Nanopore Company is qualified if more than 800 film embedding holes are formed on 2048 micropores. It can be seen that the existing film embedding hole difficulty is very high, and the chip device prepared by the preparation method of the present invention has a high proportion of effective holes.

[0023] In general, the present invention is based on a process of multi-stage centrifugal addition of film-forming materials, and designs a preparation method for a chip device based on a sandwich film-forming structure. This method not only maintains the stability of the membrane solution morphology during the chip preparation process and the cleaning process, which is beneficial to the long-term stable storage of the membrane solution, but also ensures the consistency of the membrane solution in the microporous array, thereby making the film-forming efficiency of the chip device high, and the amphiphilic molecular layer formed in the microporous array has better uniformity and higher consistency.

[0024] Specifically, the present invention uses a multi-stage centrifugation process to accurately and quantitatively control the amount of the first electrolyte, membrane solution and viscous protective liquid in each microporous structure in turn, which is beneficial to improve the success rate of film formation of the microporous array. First, the first electrolyte is convex in the first layer of micropores, so that the membrane solution will fill the lower areas on both sides, which can better support the membrane solution and is beneficial to the formation of the amphiphilic molecular layer; secondly, the membrane solution is thick at the edge and thin in the middle after polymerization, so that the final standard amphiphilic molecular layer is mainly formed in the central area of ​​the circular membrane, which is beneficial to improve the uniformity and consistency of the amphiphilic molecular layer formed in the microporous array; finally, the membrane solution and the first electrolyte are partially sealed with a viscous protective liquid, and the viscous protective liquid will not affect the chemical properties and physical form of the membrane solution (that is, through the cooperation of a specific viscous protective liquid and a local sealing scheme, the membrane solution morphology can maintain a high consistency and stability during the transportation and use of the device, which is beneficial to improve the success rate of subsequent film formation and the consistency of the membrane). In addition, the viscous protective liquid has low toxicity and is friendly to production personnel.

[0025] In addition, the sandwich film-forming structure formed by the first electrolyte, the membrane solution and the viscous protective solution is also conducive to further maintaining the good morphology of the membrane solution during long-term storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0027] Figure 1 is a schematic diagram of a chip device in an exemplary embodiment of the present invention; Figure 2 A schematic diagram of a two-layer microporous structure prepared in an exemplary embodiment of the present invention; Figure 3 is a schematic diagram of an uncured gel electrolyte on a microporous structure in an exemplary embodiment of the present invention; Figure 4 A schematic diagram of a heated gel electrolyte flowing into a microporous structure in an exemplary embodiment of the present invention; Figure 5 A schematic diagram of the morphology of the gel electrolyte in the first layer of micropores in an exemplary embodiment of the present invention; Figure 6Schematic diagram of a gel electrolyte and a polymerized membrane solution solidified in micropores in an exemplary embodiment of the present invention; Figure 7a A schematic diagram of a sandwich structure formed by a viscous protective solution, a membrane solution and a gel electrolyte in an exemplary embodiment of the present invention; Figure 7b is a schematic diagram of an angle α formed by a membrane solution and a viscous protective liquid in an exemplary embodiment of the present invention; Figure 7c A schematic diagram of another double-layer microporous structure in an exemplary embodiment of the present invention; Figure 8 FIG. 1 is an electrical characterization diagram of a chip device after film formation in an exemplary embodiment of the present invention.

[0028] Summary of reference numerals: 1. base layer; 2. microporous structure, 21. first layer of micropores, 22. second layer of micropores; 3. film-forming unit; 4. first electrolyte; 5. membrane solution; 6. viscous protective liquid; 7. cover layer; 8. flow channel structure, 81. first opening, 82. second opening. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] Herein, suffixes such as "module", "component" or "unit" used to represent elements are only used to facilitate the description of the present invention, and have no specific meanings by themselves. Therefore, "module", "component" or "unit" can be used mixedly.

[0031] In this document, the terms "upper", "lower", "inner", "outer", "front", "back", "one end", "the other end" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0032] In this document, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, an indirect connection through an intermediate medium, or a connection between the two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. "And / or" in this document includes any and all combinations of one or more of the listed related items.

[0033] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0034] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0035] As used in this specification, the term "about" typically means + / - 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, and even more typically + / - 0.5% of the stated value.

[0036] As used in this specification, the term "same" or "approximately the same" typically means + / -5% of the height or the content, more typically + / -4% of the height or the content, more typically + / -3% of the height or the content, more typically + / -2% of the height or the content, even more typically + / -1% of the height or the content, even more typically + / -0.5% of the height or the content.

[0037] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed 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 this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0038] Example 1 The present invention provides a method for preparing a chip device, and the specific preparation method is as follows: S1, providing a substrate layer, the substrate layer having a surface, and a plurality of microporous structures formed on the surface, and correspondingly the microporous structures include: being concave inwardly in a direction away from the surface to sequentially form a second layer of micropores 22 and a first layer of micropores 21, and the diameter of the second layer of micropores 22 is larger than the diameter of the first layer of micropores 21, so that a step is formed at the second layer of micropores 22 adjacent to the first layer of micropores 21, and the step is used to support a membrane solution; S2, adding a first electrolyte into the microporous structure, and performing a first centrifugation at a first speed to remove excess of the first electrolyte, and allowing the remaining first electrolyte to fill the first layer of micropores 21; under the action of centrifugation, the heights of the first electrolyte at multiple locations are the same or approximately the same; the first electrolyte is a gel electrolyte; S3, adding the membrane solution into the microporous structure so that the membrane solution fills the first layer of micropores 21 or the membrane solution overflows the first layer of micropores 21, performing a second centrifugation at a second speed, and then 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 contents of the membrane solution at multiple locations are the same or approximately the same; S4, adding a viscous protective liquid into the microporous structure, and performing a third centrifugation at a third speed so that the viscous protective liquid fills the second layer of micropores 22, and finally the viscous protective liquid, the membrane solution and the gel electrolyte form a stable sandwich structure in the microporous structure.

[0039] The sandwich layer structure can not only maintain the good morphology of the membrane solution in the micropores during the chip preparation process and the cleaning process, but also maintain the consistency of the membrane solution in the micropore array.

[0040] Preferably, the present invention uses a multi-stage centrifugal process to form a sandwich layer structure on the base layer. On the one hand, the centrifugal process is used in conjunction with a special microporous structure (which is designed to be larger at the top and smaller at the bottom) to accurately arrange the content and shape of each layer structure of the sandwich layer, and at the same time, a viscous protective liquid is used to seal the membrane solution in a local area (i.e., within the microporous structure), which can not only improve the stability of the sandwich layer structure, but also simplify the subsequent processing difficulty (such as the base layer and the cover layer will not affect the stability of the sandwich layer structure during the coordinated processing process).

[0041] Preferably, the first electrolyte is in the form of a protrusion in the first layer of micropores, and the highest point of the protrusion is on the same horizontal plane as the first end of the first layer of micropores.

[0042] Preferably, in the sandwich layer structure, the first part of the membrane solution covering the step has a first liquid level, and the second part of the membrane solution filling the gap has a second liquid level, and the first liquid level and the second liquid level are in contact with the viscous protective liquid so that the first liquid level and the second liquid level form an angle α.

[0043] In some embodiments, the angle α is about 115-125°. Applicants note that when an inverted trapezoidal microporous structure is used, the support provided by the second layer of micropores is particularly conducive to the two liquid surfaces of the membrane solution forming an angle of about 115-125° (preferably 120°).

[0044] Among them, the first electrolyte, membrane solution, and viscous protective liquid can form a stable form with lower energy within this specific angle range (in other words, at this time, under the dual squeezing of the first electrolyte and the viscous protective liquid, the cross-section of the liquid surface where the membrane solution contacts the viscous protective liquid forms a geometric figure similar to half of a regular hexagon. At this time, the forces between the liquid surfaces are more balanced and the system energy is also lower).

[0045] Of course, in other embodiments, a local geometric figure similar to a pentagon may be formed between the first liquid surface and the second liquid surface (such as several liquid surfaces where the membrane solution contacts the viscous protective liquid, such as the first liquid surface (such as Figure 7b left side), the second level, the first level (as shown in Figure 7b The liquid surface shown on the right is close to the three adjacent sides of the pentagon. For example, the angle α can also be about 103-113°. At this time, the membrane solution can still maintain a relatively stable form.

[0046] Further, in some embodiments, the angle α (such as Figure 7b The value of angle (as shown) can also be about 98-130° to enable the membrane solution to form a stable form with lower energy.

[0047] Furthermore, the viscous protective liquid includes a water-soluble high molecular polymer and / or a water-soluble non-ionic high molecular compound.

[0048] Specifically, the water-soluble high molecular polymer includes polyacrylic acid (PAA).

[0049] Specifically, the water-soluble nonionic polymer compound includes polyvinyl pyrrolidone (PVP).

[0050] Furthermore, the polyvinyl pyrrolidone (PVP) includes PVP K30 and / or PVP K60. The electrical characterization diagram of the chip device prepared based on PVP K30 after film formation is as follows: Figure 8 shown.

[0051] In some embodiments, the first layer of micropores 21 has a structural thickness of 60-150 μm and a diameter of 70-120 μm; the second layer of micropores 22 has a structural thickness of 20-50 μm and a diameter of 90-150 μm.

[0052] Preferably, the first electrolyte is viscous, and the gel component in the first electrolyte is methacryloyl gelatin (GelMA) or methacryloyl hyaluronic acid gelatin (HAMA), and the concentration range of the gel component is 5-15%.

[0053] Furthermore, the solute of the membrane solution is phospholipid, artificial synthetic polymer membrane, or a mixture of phospholipid and polymer membrane; the solvent of the membrane solution is a non-polar solvent, and the non-polar solvent includes AR20, silicone oil, hexadecane, decane or a mixture thereof.

[0054] In addition, the preparation method further comprises cutting the base layer and assembling a flow channel structure using a cover layer.

[0055] The present invention also provides a film forming method, which comprises: A chip device is provided, wherein a flow channel structure is formed in the chip device, and the chip device is prepared by the preparation method as described above; Injecting a cleaning liquid (such as water) into the flow channel structure at least once, so that the cleaning liquid enters the second layer of micropores to dilute the viscous protective liquid; 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 (such as amphiphilic molecules) in the membrane solution automatically arranges to form an amphiphilic molecule layer under the action of the first electrolyte and the second electrolyte.

[0056] In some embodiments, the cleaning solution may also be an electrolyte.

[0057] The following embodiment provides an example of a specific method for preparing a chip device.

[0058] (1) Microporous structure 2 (such as Figure 2 As shown in FIG. 1 ), the micropore structure 2 includes a second layer of micropores 22 and a first layer of micropores 21 which are recessed inwardly in a direction away from the surface of the silicon wafer and are formed in sequence.

[0059] Preferably, the first layer of micropores 21 has a thickness ranging from 60 to 150 μm and a diameter ranging from 70 to 120 μm; the second layer of micropores 22 has a thickness ranging from 20 to 50 μm and a diameter ranging from 90 to 150 μm.

[0060] The diameter of the second layer micropores 22 is greater than that of the first layer micropores 21 , so that steps are formed at the second layer micropores 22 adjacent to the first layer micropores 21 , which helps to stably support the membrane solution added subsequently.

[0061] See also Figure 7b As shown, the double-layer microporous structure proposed in the present invention can enable the membrane solution to be stored in a low-energy stable form. Specifically, the membrane solution covers the first part of the step to form a first liquid surface, and the membrane solution fills the second part of the gap to form 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 angle α in the range of 98-130°, so that the membrane solution can polymerize to form a low-energy stable form.

[0062] In another exemplary embodiment of the present invention, see Figure 7c As shown, the cross-section of the first layer of micropores is square; the cross-section of the second layer of micropores is an inverted trapezoid. This structure of larger top and smaller bottom can form an angle α of approximately 115-125°, and can also enhance the support for the membrane solution, ensuring that the membrane solution remains in a low-energy stable state during long-term transmission.

[0063] In some embodiments, a SUEX or AUEX photoresist dry film may be directly laminated on a silicon wafer (ie, a base layer) to prepare a two-layer microporous structure.

[0064] In some other embodiments, the desired two-layer microporous structure may be embossed on a special embossing adhesive by heating it to about 1200° C., and then demolding may be performed after the temperature drops.

[0065] (2) Gel electrolyte 4 (i.e., first electrolyte) is dripped into the microporous structure 2. Initially, the uncured gel electrolyte has a relatively high viscosity and cannot enter the microporous structure (e.g., Figure 3 As shown in FIG. 1 ), after the silicon wafer is placed on a hot plate and heated to about 90°C, the viscosity of the uncured gel electrolyte 4 decreases and flows into the microporous structure, as shown in FIG. Figure 4 shown.

[0066] Then, the silicon wafer is placed on a spinning machine and spun at a speed of 500-1000 rpm for 2-10 minutes to remove excess uncured gel electrolyte on the surface and inside of the microporous structure under the action of centrifugal force, thereby maintaining the consistency of the gel electrolyte liquid level in the microporous array, so that the remaining gel electrolyte fills the first layer of micropores 21.

[0067] In other words, the height of the liquid level of the gel electrolyte can be adjusted by adjusting the rotation speed. The higher the rotation speed, the more gel is thrown away by the centrifugal force, and the lower the liquid level.

[0068] After the spinning is completed, the gel electrolyte is allowed to stand for 5 minutes and then cooled to room temperature naturally. Since the inner wall material of the micropores is hydrophobic, the gel electrolyte presents a convex shape in the first layer of micropores (such as Figure 5 As shown), and the highest point of the protrusion is on the same horizontal plane as the first end of the first layer of microholes.

[0069] Use a UV curing light source with a wavelength of 365 nm to cure the silicon wafer at a height of 5 cm for 30 seconds. The cured gel electrolyte can better support the membrane solution, and the liquid level of the gel electrolyte in each microwell is consistent.

[0070] Preferably, the gel component in the gel electrolyte is methacryloyl gelatin (GelMA) or methacryloyl hyaluronic acid gelatin (HAMA), and the concentration range is 5-15%.

[0071] (3) preparing a membrane solution 5, wherein the membrane-forming material of the membrane solution 5 includes phospholipids, artificial synthetic polymer membranes, or a mixture of phospholipids and polymer membranes.

[0072] The film-forming material can be dissolved in AR20, silicone oil, hexadecane, decane or a mixture thereof.

[0073] (4) Add the membrane solution 5 into the microporous structure 2. The membrane solution will first fill the lower part of the first layer of micropores 21 to facilitate the formation of the membrane, and finally fill the first layer of micropores 21 or overflow the first layer of micropores 21. Place the silicon wafer on a spinning machine and use centrifugal force at a speed of 100-300 rpm to remove excess membrane solution. Under the centrifugal effect, the content of the membrane solution in the microporous array is kept the same or approximately the same. At this time, a small amount of membrane solution 5 will be on the side wall of the micropore.

[0074] Furthermore, the substrate layer with the membrane solution added thereto may be refrigerated at a temperature of 2-8°C. For example, the silicon wafer may be placed in a refrigerator at 4°C for 30 minutes, and the membrane solution will slowly polymerize and spread on the gel electrolyte 4 in the micropores (e.g. Figure 6 as shown).

[0075] (5) Adding a viscous protective liquid 6 into the microporous structure, removing excess of the viscous protective liquid by centrifugation, so that the viscous protective liquid fills the second layer of micropores, and finally the viscous protective liquid, the membrane solution and the first electrolyte form a stable sandwich layer structure (i.e., a sandwich membrane structure, such as Figure 7a as shown).

[0076] Furthermore, the viscous protective liquid includes polyacrylic acid (PAA) and / or polyvinyl pyrrolidone (PVP).

[0077] Furthermore, the viscous protective liquid is preferably PVP K30. The PVP K30 solution is dripped onto the silicon wafer and spun at a speed of 300-500 rpm for 15 minutes using a spun glue machine to remove excess PVP K30. The effect is as follows: Figure 7a shown.

[0078] 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 mixing beaker can be placed on a shaker and shaken at 100 rpm at room temperature for 1 hour until it is completely dissolved. Alternatively, a 30% by mass PVP K30 solution can be directly purchased.

[0079] Of course, in other embodiments, the user may also select solutions of different qualities according to different application environments, different film-forming materials, and different film-forming standards.

[0080] (6) Cut the silicon wafer, use waterproof gaskets, clamps, and screws to assemble the flow channel structure to form a device such as Figure 1 shown.

[0081] After the device is assembled, a cleaning solution and a second electrolyte are sequentially injected into the flow channel to form a membrane.

[0082] In some embodiments, the cleaning solution is pure water, and 300 μL of pure water is injected into the flow channel, at which point the PVP K30 will be diluted by the pure water. Every 5 minutes, use 1000 μL of pure water to slowly rinse the flow channel, and repeat three times. The fourth time is to pass 300 μL of protein-containing electrolyte, at which point the PVP K30 aqueous solution is completely rinsed with pure water, and the membrane solution just forms an amphiphilic molecular membrane (or, amphiphilic molecular layer, also referred to as "membrane") between the gel electrolyte and the protein-containing electrolyte, and the protein can be normally embedded in the amphiphilic molecular membrane to form a channel on the membrane.

[0083] DNA passes through the protein channel under the action of the electric field. Depending on how much the channel is blocked, the current at both ends of the membrane changes when voltage is applied. Based on the change in current, we can know which sequences of DNA have passed through the channel, thereby achieving sequencing.

[0084] The film-forming effect can be detected by electrical characterization of the membrane capacitance. The thickness of the membrane formed in each micropore can be determined by measuring its capacitance. The thickness of the amphiphilic molecule layer formed is different, and the electrical characterization will be different. The micropore array of the present invention has 1024 micropores, which are divided into 4 areas, AREA1 to AREA4, and the number of membrane capacitances that can be measured each time is 256. The present invention selects each AREA to measure all 1024 micropores in steps. Figure 8 Each rectangular block in the figure represents a membrane capacitor corresponding to a microporous structure, and the value in the rectangular block represents the capacitance value of the membrane capacitor.

[0085] In addition, the color depth of each rectangular electrical representation in the instrument has a certain relationship with the size of the membrane capacitance value. The value of the capacitance value can represent the state of different micropores, such as whether a film is formed and the thickness of the film layer. Specifically: the capacitance value is less than 40 pF and is displayed in green, indicating that the thickness of the amphiphilic molecular film is too large; the capacitance value is displayed in light gray within the range of 40-70 pF, indicating that the thickness of the amphiphilic molecular film is appropriate; the capacitance value is greater than 70 pF and is displayed in red, indicating that the thickness of the amphiphilic molecular film is too small.

[0086] Select a different AREA on the right, and the 256 micropores of the AREA will be enlarged and displayed on the left to facilitate reading of the specific membrane capacitance value. Figure 8 As shown, the film formation results show that the membrane capacitance in the 1024 micropores is in the range of 40-70 pF, indicating that an amphiphilic molecular membrane of suitable thickness has been formed, which is conducive to subsequent conventional embedding.

[0087] The results show that the preparation method of the present invention greatly improves the success rate of film formation on multi-channel micropores. Using membrane capacitance detection, all micropores are film-formed, the film consistency is very good, the embedded pore rate is significantly increased to more than 90%, and the effective pore ratio is greatly increased.

[0088] Furthermore, in order to verify the film forming efficiency of the present invention, a comprehensive evaluation of 20 staff members on different viscous protective solutions was collected, and the evaluation contents included: protective effect, operating efficiency, operating difficulty, and the most important membrane capacitance value, which can determine the thickness of the membrane. The results are shown in Table 1, where the protective effect is reflected in the stability of the protective film solution morphology during the chip assembly and cleaning stages, the operating efficiency is reflected in the stage of introducing different reagents, and the operating difficulty is reflected in the protection of the operator when operating the reagent and the impact on the environment afterwards.

[0089] Table 1 Comprehensive evaluation results of staff on protective fluids with different viscosities As can be seen from Table 1, the use of protective liquid materials such as PAA and PVP significantly improves the final film-forming pore embedding rate; it is worth noting that in the film-forming device launched by Oxford Nanopore, more than 800 film-forming pores on 2048 micropores are considered qualified, that is, the embedding success rate reaches 39%. It can be seen that the existing film-forming process is very difficult and the embedding success rate is very low. On the contrary, the multi-stage centrifugal process, special micropore design and special protective liquid used in the present invention unexpectedly directly increase the embedding success rate to about 88%.

[0090] Furthermore, PVP directly increases the embedding success rate to more than 91%, and the operating efficiency and difficulty meet the application requirements. In particular, PVP K30 can achieve a 100% embedding success rate under optimal conditions, with low operating difficulty and high operating efficiency.

[0091] The present invention innovatively uses a spinning process (i.e., a multi-stage centrifugation process) to add film-forming materials. In view of the fact that the chips currently used in the field of nanopore sequencing are first processed by micro-nano processing and then film-embedded holes are formed through complex microfluidic operations and multiple reagents, the present invention creatively integrates the film-forming materials into the chip manufacturing process through micro-nano processing, and then the film formation only requires pure water to be passed into the flow channel. The film formation success rate is above 90% (even above 95%, such as 100%), and the hole embedding success rate can reach 90% (even above 95%, such as 100%).

[0092] Example 2 Based on the preparation method of Example 1, the present invention provides a chip device, comprising: A base layer 1, wherein at least one microporous structure 2 is provided on the base layer 1; The microporous structure 2 comprises a first layer of micropores 21 and a second layer of micropores 22, wherein the second layer of micropores 22 is located on the upper layer of the first layer of micropores 21; For example, the base layer has a surface, and a plurality of microporous structures are formed on the surface, and the microporous structures correspondingly include: being recessed inwardly in a direction away from the surface to sequentially form a second layer of micropores 22 and a first layer of micropores 21, and the diameter of the second layer of micropores 22 is larger than the diameter of the first layer of micropores 21, so that a step is formed at the second layer of micropores 22 adjacent to the first layer of micropores 21, and the step is used to support the membrane solution.

[0093] For example, in some embodiments, the diameter of the second end of the second layer of micropores is greater than the diameter of the first layer of micropores, in which case the plane formed by the connection between the second layer of micropores and the first layer of micropores is the step. Specifically, the cross section of the second layer of micropores (i.e., the plane perpendicular to the surface) can be rectangular or approximately rectangular.

[0094] For another example, in some embodiments, the diameter of the second layer of micropores gradually decreases from the first end to the second end, and correspondingly, the second end of the second layer of micropores forms the step (ie, slope) to support the membrane solution.

[0095] In other words, in this embodiment, the cross section of the second layer of micropores is an inverted trapezoidal structure with a larger top and a smaller bottom. This inverted trapezoidal design is conducive to the two liquid surfaces of the membrane solution forming an angle α of about 115-125° (preferably 120°) at the step (e.g. Figure 7b At this time, the first electrolyte, the membrane solution and the viscous protective solution can form a stable form with lower energy.

[0096] In other embodiments, a local geometric figure similar to a pentagon can be formed between the two sides where the membrane solution contacts the viscous protective liquid (such as the first liquid surface on both sides) and the liquid surface formed by the membrane solution filling the first layer of micropores (such as the second liquid surface), and the angle α formed by them is approximately 103-113°.

[0097] Furthermore, it also includes: a film forming unit 3, the film forming unit 3 includes: a first electrolyte 4, a film solution 5 and a viscous protective solution 6, wherein: The first electrolyte 4 is in the first layer of micropores 21, and after solidification, it presents a convex shape, which can better support the membrane solution; the membrane 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 micropores 22, and the membrane solution 5 is between the viscous protective liquid 6 and the first electrolyte 4, and the viscous protective liquid 6, the membrane solution 5 and the first electrolyte 4 form a stable sandwich layer structure, maintaining the morphology of the membrane solution 5 in the micropores and the morphological consistency in the micropore array; The chip device further includes a cover layer 7 , and the cover layer 7 and the base layer 1 are assembled into a flow channel structure 8 , and the flow channel structure 8 is respectively provided with a first opening 81 and a second opening 82 .

[0098] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above-mentioned embodiment methods can be implemented by means of software plus a necessary general hardware platform, and of course by hardware, but in many cases the former is a better implementation method. Based on such an understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, can be embodied in the form of a software product, which is stored in a storage medium (such as ROM / RAM, a magnetic disk, or an optical disk), and includes a number of instructions for a computer terminal (which can be a mobile phone, a computer, a server, or a network device, etc.) to execute the methods described in each embodiment of the present invention.

[0099] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0100] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for preparing a chip device, characterized in that: The chip device comprises: a base layer, and correspondingly, the preparation method comprises: S1, providing a substrate layer, the substrate layer having a surface, and a plurality of microporous structures formed on the surface, the microporous structures correspondingly comprising: a second layer of micropores (22) and a first layer of micropores (21) which are sequentially formed by being recessed inwardly 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), so that steps are formed at the second layer of micropores (22) adjacent to the first layer of micropores (21), and the steps are used to support a membrane solution; S2, adding a first electrolyte into the microporous structure, and performing a first centrifugation at a first speed to remove excess first electrolyte and allow the remaining first electrolyte to fill the first layer of micropores (21); under the action of centrifugation, the heights of the first electrolyte at multiple locations are the same or approximately the same; the first electrolyte is a gel electrolyte; S3, adding the membrane solution into the microporous structure so that the membrane solution fills the first layer of micropores (21) or the membrane solution overflows the first layer of micropores (21), performing a second centrifugation at a second speed, and then polymerizing the membrane solution and filling the gap between the first electrolyte and the first layer of micropores (21), and partially covering the step; under the action of centrifugation, the contents of the membrane solution at multiple locations are the same or approximately the same; S4, adding a viscous protective liquid into the microporous structure, and performing a third centrifugation at a third speed so that the viscous protective liquid fills the second layer of micropores (22), and finally the viscous protective liquid, the membrane solution and the first electrolyte form a stable sandwich layer structure in the microporous structure.

2. The preparation method according to claim 1, characterized in that 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; or, 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.

3. The preparation method according to claim 1, characterized in that: In the sandwich layer structure, the first part of the membrane solution covering the step has a first liquid level, and the second part of the membrane solution filling the gap has a second liquid level. The first liquid level and the second liquid level are in contact with the viscous protective liquid so that the first liquid level and the second liquid level form an angle α in the range of 98°-130°.

4. The preparation method according to claim 3, characterized in that: The angle α is 103°-113° or 115°-125°.

5. The preparation method according to claim 1, characterized in that: The viscous protective liquid includes a water-soluble high molecular polymer and / or a water-soluble non-ionic high molecular compound.

6. The preparation method according to claim 5, characterized in that: The water-soluble high molecular polymer includes polyacrylic acid; and / or the water-soluble non-ionic high molecular compound includes polyvinyl pyrrolidone.

7. The preparation method according to claim 6, characterized in that The polyvinyl pyrrolidone includes PVP K30 and / or PVP K60.

8. 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, and the concentration range of the gel component is 5-15%.

9. The preparation method according to claim 1, characterized in that: The solute of the membrane solution includes phospholipids, artificial synthetic polymer membranes or a mixture of phospholipids and polymer membranes; the solvent of the membrane solution is a non-polar solvent, and the non-polar solvent includes AR20, silicone oil, hexadecane, decane or a mixture thereof; or, the preparation method further includes cutting the base layer and assembling a flow channel structure using a cover layer.

10. A film forming method, characterized in that: The film forming method comprises: Provided is a chip device, wherein a flow channel structure is formed in the chip device, and the chip device is prepared by any one of the preparation methods of claims 1 to 9; Injecting a cleaning liquid into the flow channel structure at least once, so that the cleaning liquid enters the second layer of micropores to dilute the viscous protective liquid; 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.

Images