Nanopore sequencing chip, preparation method and packaging structure thereof

Abstract

The application discloses a nanopore sequencing chip, a preparation method thereof and a packaging structure, and relates to the fields of micro-nano processing, advanced packaging and biological medicine. The nanopore sequencing chip comprises a first insulating layer, a second insulating layer and an electrode, the second insulating layer is arranged on the first insulating layer, a plurality of holes arranged in an array are arranged on the second insulating layer, the holes penetrate through the second insulating layer and are in communication with the first insulating layer, the electrode is arranged at the bottom of the holes, a hydrophobic area is arranged on the second insulating layer, the hydrophobic area is located above the holes and is arranged around the holes, and a hydrophilic modified area is arranged at the bottom of the holes. The preparation method is simple, the light diffraction characteristics can be utilized, and a double-layer structure can be prepared by only one photoetching exposure technology. The packaging structure adopts a metal lead and a leadless packaging form, the crosstalk problem of the metal lead is solved, and separate transportation is adopted, so that the shelf life is prolonged. The nanopore sequencing chip has the advantages of simple structure, low cost and good stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of micro-nano processing, advanced packaging and biomedical treatment, and in particular to a nanopore sequencing chip, a preparation method thereof and a packaging structure. BACKGROUND

[0002] The nanopore sequencing technology has the characteristics of long read length, low cost, high throughput and high integration, and has a wide application in the biological field of molecular detection and gene sequencing.

[0003] The basic working principle of the nanopore sequencing technology is that in a cavity filled with electrolyte, an insulating impermeable film with nanoscale pores divides the cavity into two small chambers. When a voltage is applied to the electrolyte chamber, ions or other charged molecular substances can pass through the small pores to form a stable detectable ion current. When the molecules to be detected pass through the nanometer pore driven by the electric field force, it will cause the timing change of the ion current signal, so that the information of the analyte, including the sequence information of the nucleic acid molecule, can be analyzed from the continuous change of the ion current signal.

[0004] The nanopore sequencing chip includes a photoresist layer and a pore array arranged on the photoresist layer. Each individual pore in the pore array is an independent sequencing unit. Therefore, the number and stability of the thin film formed in the pore array on a single chip are crucial to the throughput and quality of the detection. At the same time, the cost of the nanopore sequencing chip is directly related to the end price of the nanopore sequencing application, and thus affects the market promotion of the product. The current nanopore sequencing chip is basically a double-layer structure, such as some structures disclosed by Oxford Nanopore Technologies in patent CN115803624A. These structures have complex shapes, high processing costs and great difficulty. In addition, due to the small pore size and the hydrophobicity of the photoresist layer, these characteristics cause the hydrophobicity of the pore bottom, and it is difficult for the ion solution to infiltrate the pore bottom and conduct with the electrode, resulting in poor pore infiltration and low conduction rate, thereby affecting the film formation rate and the stability of the film after film formation. SUMMARY

[0005] In order to solve the problems existing in the prior art, the purpose of the present application is to provide a nanopore sequencing chip, a preparation method thereof and a packaging structure. The nanopore sequencing chip of the present application has low cost, high yield, good structure infiltration, high conduction rate, simple preparation method, fast film formation and good stability of the film after film formation.

[0006] The present application provides the following technical solutions:

[0007] In a first aspect, the present application provides a nanopore sequencing chip, comprising a first insulating layer, a second insulating layer and an electrode, wherein the second insulating layer is arranged on the first insulating layer, and a plurality of arrayed pores are arranged on the second insulating layer; the pores penetrate through the second insulating layer and communicate with the first insulating layer; the electrode is arranged at the bottom of the pores; a hydrophobic region is arranged on the second insulating layer, and the hydrophobic region is arranged above each pore and surrounds each pore, and the hydrophobic region is conducive to the generation, fixation and storage of organic solvents of a membrane; the second insulating layer and the hydrophobic region form a double-layer-like insulating layer pore sequence structure; a hydrophilic modification region is arranged at the bottom of the pores; the hydrophilic modification region is obtained by modification with a hydrophilic modification solution, and the hydrophilic modification solution is selected from one of a chitosan solution, a dopamine solution and a polylysine solution.

[0008] Further, the arrayed pores are separated from each other by a second insulating layer medium, and the shape of the pores can be circular or irregular.

[0009] Further, the shape of the electrode can be circular or polygonal, and the geometric center of the electrode coincides with the geometric center of the shape of the pores on the second insulating layer.

[0010] Further, the hydrophobic region is an oil storage region and / or a hydrophobic modification region. The oil storage region is obtained by forming a surface microstructure on the surface of the second insulating layer; and the hydrophobic modification region is obtained by hydrophobic modification at the top of the pores.

[0011] Further, the nanopore sequencing chip further comprises a signal transmission component connected with the electrode for transmitting a test signal.

[0012] Further, the signal transmission component comprises an electrode line and a chip pad, and the chip pad is arranged outside the first insulating layer or the second insulating layer, and the chip pad is connected with the electrode through the electrode line.

[0013] Preferably, the hydrophobic region comprises an oil storage region. The oil storage region can form a double-layer-like insulating layer structure with the second insulating layer, and by designing a honeycomb-like double-layer-like insulating layer structure, the stress generated by photoresist during micro-nano processing can be reduced, the setting of the peripheral oil storage region on the chip can be cancelled, the chip area can be reduced, and the manufacturing cost of the chip can be reduced.

[0014] Preferably, the distance between the edge of the oil storage region and the edge of the pore is 3-50 μm.

[0015] Preferably, the height of the oil storage region is 1-50 μm.

[0016] Preferably, the first insulating layer is a single crystal silicon layer or a glass layer.

[0017] Preferably, the material of the second insulating layer is hydrophobic; the second insulating layer is prepared from a photosensitive insulating material. Preferably, the photosensitive material is selected from one of the following: photoresist, UV glue, and photoresist film.

[0018] Preferably, the surface of the second insulating layer is provided with a plurality of convex structures or a plurality of concave structures, and the spaces between the plurality of convex structures or the plurality of concave structures form oil storage areas. The second insulating layer and the plurality of convex structures or the plurality of concave structures on the surface thereof form a double-layer-like insulating layer hole structure that can improve film stability.

[0019] Further, the convex structures can be circular, circular-like, or polygonal, and the polygonal structures include regular polygonal structures and irregular polygonal structures.

[0020] Preferably, the convex structures are hexagonal, and a plurality of the hexagonal convex structures are regularly arranged on the second insulating layer. Preferably, the side length of the hexagonal convex structures is 1 μm to 50 μm.

[0021] Preferably, regular flow channels are formed between the plurality of hexagonal convex structures, and the regular flow channels and the hexagonal convex structures form a honeycomb structure. Preferably, the depth of the regular flow channels is 1 μm to 50 μm.

[0022] Further, the concave structures can be circular or irregular polygonal, and a plurality of the concave structures are regularly arranged to form oil storage areas. Preferably, the depth of the concave structures is 1 μm to 50 μm.

[0023] Preferably, the holes are circular holes, the diameter of the holes is 20 μm to 150 μm, and the height of the holes is 10 μm to 150 μm.

[0024] Preferably, the hydrophobic area includes a hydrophobic modified area, the hydrophobic modified area is obtained by hydrophobic modification using a hydrophobic modification solution, and the hydrophobic modification solution is selected from one of the following: a fluorine-containing compound, a polysilicon compound, and a synthetic carbon chain compound.

[0025] Preferably, the mass concentration of the hydrophobic modification solution is 10% to 60%.

[0026] Further, the hydrophobic modification includes the following steps:

[0027] The hydrophobic modification solution is added to the geometric center of the surface of the second insulating layer, the hydrophobic modification solution spreads to the surrounding area, covers the upper surface of the second insulating layer and the upper end of the holes, and does not penetrate into the bottom of the holes; then the temperature is heated to 100°C to 200°C for 30 min to 180 min to form a hydrophobic area above the holes.

[0028] Preferably, the volume of the hydrophobic modification solution is 0.5-5 μL.

[0029] Preferably, the hydrophobic modification solution is a fluorine-containing compound, and the hydrophobic modification comprises the following steps:

[0030] The fluorine-containing compound is dropped into a heating device, and the fluorine-containing compound is evaporated into a gaseous state under high temperature. The gaseous fluorine-containing compound is driven by high-purity nitrogen or inert gas to fill the modification environment and uniformly deposit on the surface of the second insulating layer to form a hydrophobic region.

[0031] The modification environment is composed of a closed transparent cavity, the top of the cavity is provided with an air inlet and an exhaust hole, and the opposite surface of the air inlet is provided with a heating device and a sample bearing device.

[0032] During the modification process, the sample bearing device uniformly drives the sample to be modified to rotate, the air inlet continuously supplies heated nitrogen or inert gas, and the heating device can rapidly control temperature change.

[0033] Further, due to the small pore size of the hole and the hydrophobic material of the photoresist, it is difficult for the polar liquid to infiltrate to the bottom of the hole, and usually vacuum or other gas displacement methods are required for infiltration and conduction. For example, Chengdu Qicang Technology Co., Ltd. discloses a technology scheme for using carbon dioxide displacement technology to conduct the hole in the patent CN215757392U. In addition, hydrophilic modification of the bottom of the hole can enable the nanopore sequencing chip to be infiltrated and conducted without vacuum or other gas displacement methods, but it is difficult to partially hydrophilically modify the hole.

[0034] Therefore, the present application provides a method for preparing a hydrophilic modification region in the above nanopore sequencing chip, comprising the following steps:

[0035] S1, hydrophilic modification; a hydrophilic modification solution with a mass concentration of 5%-20% is used to infiltrate the hole, and the modification is carried out at a temperature of 20-50℃ for 30-180min. If the concentration of the hydrophilic modification solution is greater than the range of the present application, the solution modification is easy to appear flocculent precipitate on the surface of the chip, which increases the difficulty of subsequent cleaning and affects the infiltration effect in the hole, thereby affecting the modification effect.

[0036] S2, remove the hydrophilic modification solution from the hole opening and retain the hydrophilic modification solution at the bottom of the hole, so that the upper end of the hole returns to a hydrophobic state; then age it for more than 5 days, so that the bottom of the hole forms a hydrophilic modification region under the action of the hydrophilic modification solution.

[0037] Preferably, the hydrophilic modification solution is a chitosan solution, which has high modification stability, long-lasting effect, and strong adhesion to the hydrophobic polymer material. The solvent of the chitosan solution is an acetic acid solution, which is prepared by diluting 1 mol / L acetic acid with pure water by 100 times.

[0038] Further, the specific steps for removing the hydrophilic modification solution from the orifice part and retaining the hydrophilic modification solution at the bottom of the hole are as follows:

[0039] The hole sequence structure is washed with pure water and dried, and then is rotated at a uniform speed under vacuum adsorption conditions and the surface thereof is washed with a high-pressure water gun until the hydrophilic modification solution on the upper part of the hole and the surface of the second insulating layer is washed clean and the hydrophilic modification solution remains at the bottom of the hole; the water on the surface thereof is blown dry with an air gun, so that the moisture on the surface of the second insulating layer is removed and the hole remains in a wet state.

[0040] Preferably, the angle between the muzzle of the high-pressure water gun and the surface thereof is 15°-60°; and the angle between the muzzle of the air gun and the surface thereof is maintained at 15°-60°.

[0041] Further, in the step S2, after aging, the chip is washed again with a high-pressure water gun to remove the crystal, and then is blown dry and heated to dry the moisture.

[0042] In a second aspect, the application further provides another nanopore sequencing chip, which is based on the nanopore sequencing chip in the first aspect, and the signal transmission component of the nanopore sequencing chip further comprises an integrated circuit area and a first electrode line, the integrated circuit area is arranged on the first insulating layer and is connected with the electrode through the first electrode line. One end of the electrode line is connected with the chip pad, and the other end is connected with the electrode through the integrated circuit area and the first electrode line.

[0043] Further, the first insulating layer comprises a substrate layer and an insulating layer, the insulating layer is arranged between the second insulating layer and the substrate layer; and the integrated circuit area is located between the substrate layer and the insulating layer.

[0044] Preferably, the electrode is arranged on the substrate layer and communicates with the outside of the first insulating layer, and the first electrode line is arranged in the inside of the substrate layer and connects the electrode and the integrated circuit area.

[0045] Further, the plurality of array-arranged holes on the second insulating layer form a hole sequencing area, and the integrated circuit area is arranged on the side of the hole sequencing area.

[0046] Further, the substrate layer comprises a single crystal silicon layer, and the integrated circuit area is arranged between the single crystal silicon layer and the insulating layer.

[0047] Preferably, the substrate layer further comprises a glass layer, and the single crystal silicon layer is located between the glass layer and the insulating layer.

[0048] Preferably, the integrated circuit area comprises a signal amplifier (TIA), a multiplexer (MUX) and an analog-to-digital converter (ADC); the electrode is connected to the signal amplifier through a first electrode line; the signal amplifier, the multiplexer and the analog-to-digital converter are sequentially connected through a fourth electrode line.

[0049] Preferably, after the electrode receives a signal, the electrode can transmit a current signal to the signal amplifier through the first electrode line, the current signal is amplified by the signal amplifier and then transmitted to the multiplexer through the fourth electrode line, the multiplexer receives current signals of multiple electrodes at the same time, and then transmits the current signals to the analog-to-digital converter through the fourth electrode line, and the analog-to-digital converter converts the current signals into digital signals and then outputs the digital signals.

[0050] Preferably, the integrated circuit area is provided with a plurality of.

[0051] In a third aspect, the application further provides a preparation method of the nanopore sequencing chip according to the first aspect, which utilizes the diffraction characteristics of light to process the nanopore sequencing chip into a double-layer insulating layer-like hole structure through one-time photolithography exposure technology, the double-layer insulating layer-like honeycomb structure in the application not only forms a hydrophobic structure similar to the Wenzel-Cassie model to make the surface exhibit strong hydrophobicity, but also acts as a stress release area, thereby saving chip area and reducing cost without setting a stress release functional area on the chip; the method comprises the following steps:

[0052] The electrode is arranged on the first insulating layer, and photoresist is coated on the surface of the first insulating layer to form a photoresist surface; according to the diffraction characteristics of light, the pattern on the mask plate is transferred to the second insulating layer, and a plurality of holes corresponding to the electrode are formed in the second insulating layer, and an oil storage area is also formed; finally, the bottom of the hole is hydrophilically modified to form a hydrophilic modification area at the bottom of the hole, thereby obtaining the nanopore sequencing chip.

[0053] Further, the photoresist surface and the electrode are located on the same surface of the first insulating layer, and the light transmission area of the mask plate for forming the oil storage area is less than or equal to the structure size of the second insulating layer.

[0054] The second insulating layer is prepared by spin coating a layer of photoresist and exposing the photoresist once, and the upper layer and the lower layer of the second insulating layer present different structures, i.e., a double-layer structure.

[0055] Preferably, the nanopore sequencing chip can be further hydrophobically modified after the hydrophilic modification, so as to form a hydrophobic modification zone on the upper end of the pore and the surface of the second insulating layer. The hydrophobic modification zone can further enhance the hydrophobic performance of the second insulating layer and the top of the pore, improve the film formation rate and the stability of the film after film formation. The hydrophobic modification is achieved by adjusting the composition and dosage of the hydrophobic modification solution, so that the hydrophobic modification solution does not enter or less enters the bottom of the pore, or the hydrophobic modification solution does not physically or chemically react with the hydrophilic layer, thereby forming an asymmetric hydrophilic and hydrophobic structure in which the bottom of the pore is hydrophilic and the top of the pore and the surface of the second insulating layer are hydrophobic.

[0056] Further, the photolithography exposure technology includes preparing a mask plate with a corresponding line width and adjusting the exposure dose correspondingly.

[0057] Further, the exposure mode of the photolithography exposure can be selected from one of vacuum contact exposure, soft contact exposure, hard contact exposure and proximity exposure.

[0058] Further, when the exposure mode is proximity exposure, the distance between the bottom of the mask plate and the second insulating layer is less than or equal to 3 μm.

[0059] Further, when the exposure mode is hard contact exposure or vacuum contact exposure, the exposure dose is 1.5-3 times of the conventional exposure dose.

[0060] In addition, since the nanopore sequencing chip needs to be packaged, the existing packaging structure has the following problems: 1. The traditional nanopore sequencing chip uses a PC plastic or acrylic shell for bonding packaging, which has high cost and poor sealing performance; 2. The nanopore sequencing chip has a large size, and is bonded through a ball grid array (BGA) and a circuit board, which has high requirements for equipment, is complicated, has high cost and low yield; 3. The sequencing chip uses a protein pore, a buffer solution and a chip for centralized transportation, which has a limited shelf life, so that the shelf life of the chip is extremely short; 4. Some sequencing chips on the market need to design a stress release area on the bare chip, which has a large packaging area and increases the cost.

[0061] Therefore, the fourth aspect is to solve the above problems, and the application provides a packaging structure suitable for the nanopore sequencing chip of the first aspect, which comprises the nanopore sequencing chip of the first aspect, a first plastic packaging shell, a packaging frame and a first metal lead.

[0062] Preferably, the plastic packaging shell is provided with a first through hole, and the packaging frame is mounted at the bottom of the first through hole. The packaging frame is used to carry the nanopore sequencing chip and dissipate heat, so as to achieve the effects of temperature control and fixation.

[0063] The first through hole can be a window, and the window can have a quadrilateral, circular or irregular shape in a top view; the upper surface and lower surface of the window can be consistent or inconsistent; the side wall can be vertical or non-vertical, or a combination of vertical and non-vertical.

[0064] The window can have a cuboid, through hole, inverted truncated cone or irregular groove shape.

[0065] The nanopore sequencing chip card is arranged inside the packaging frame; a first liquid storage groove is formed between the upper end of the first through hole and the upper surface of the nanopore sequencing chip.

[0066] The signal transmission assembly is a first signal transmission assembly, which includes a second electrode line corresponding to the number of holes and a first chip pad, the first chip pad is arranged on the side surface of the nanopore sequencing chip, the second electrode line is arranged inside the nanopore sequencing chip, and the first chip pad is connected with the electrode through the second electrode line; a plurality of packaging frame pads corresponding to the first chip pad are arranged on the side surface of the packaging frame; the first metal lead is located above the nanopore sequencing chip; each first chip pad and packaging frame pad are connected through the first metal lead.

[0067] A plurality of first metal contacts are arranged on the outer side of the bottom of the plastic package shell, and the first metal contacts are used for connecting the conductive.

[0068] Further, a hole sequencing region is formed between the holes of the nanopore sequencing chip, and the hole sequencing region is completely exposed in the liquid storage groove, so as to further ensure the film forming rate of the nanopore sequencing chip.

[0069] Preferably, the first plastic package shell is formed by directly injection molding on the outer surface of the nanopore sequencing chip.

[0070] Preferably, the first plastic package shell can also be made by machining or molding.

[0071] Preferably, the material of the first plastic package shell is selected from one of organic glass, conventional plastic, and a mixture of silicon oxide and epoxy resin.

[0072] Preferably, the packaging frame is selected from one of a metal frame, a printed circuit board (PCB), a ceramic substrate, a silicon substrate and a quartz substrate.

[0073] Further, the number of first metal contacts is selected from one of 1-120; the number of first metal contacts, first chip pads and packaging frame pads corresponds to the number of holes. Preferably, the packaging frame has a rectangular or circular structure; the bottom shape of the first plastic package shell is consistent with the shape of the packaging frame.

[0074] In a fifth aspect, the present application further provides a packaging structure which can solve the above problems and is suitable for the nanopore sequencing chip of the second aspect, and the difference between the packaging structure and the packaging structure of the fourth aspect is that:

[0075] It comprises the nanopore sequencing chip of the second aspect, the first plastic sealing shell, the packaging frame and the first metal lead.

[0076] The signal transmission component is a second signal transmission component, which comprises a first electrode line, an integrated circuit area, a third electrode line and a second chip pad connected in sequence, the second chip pad is arranged on the side of the nanopore sequencing chip, the third electrode line is arranged inside the nanopore sequencing chip, and the second chip pad is connected with the integrated circuit area through the third electrode line. The second chip pad is connected with the packaging frame pad through the first metal lead. The number of the first metal contact, the second chip pad and the packaging frame pad is correspondingly arranged.

[0077] Further, the number of holes in the nanopore sequencing chip is an integer multiple of the number of second chip pads.

[0078] Further, the number of holes in the nanopore sequencing chip in the packaging structure does not need to correspond to the number of second chip pads one by one, and can be n times the number of second chip pads, wherein n is a positive integer. Therefore, the number of second chip pads required can be much less than the number of holes, so as to improve the density and number of holes on a single chip and the sequencing throughput, further reduce the volume and reduce the sequencing cost. In addition, the packaging structure can directly convert the data measured in real time through the integrated circuit area and output through the second chip pad, which can reduce the interference generated during signal transmission.

[0079] In a sixth aspect, the present application further provides another packaging structure which can solve the above problems and is suitable for the nanopore sequencing chip of the first aspect, comprising the nanopore sequencing chip of the first aspect, a second plastic sealing shell, a packaging substrate, a polymer gasket and a second metal lead.

[0080] The nanopore sequencing chip is arranged on the packaging substrate, the signal transmission component is a first signal transmission component, the first signal transmission component comprises a first chip pad and a second electrode wire, the first chip pad is arranged on the side of the nanopore sequencing chip, the second electrode wire is arranged inside the nanopore sequencing chip, and the second electrode wire is connected with the first chip pad and the electrode; the packaging substrate is arranged inside the second plastic sealing shell, and the high polymer gasket is arranged between the nanopore sequencing chip and the second plastic sealing shell. The upper shell is provided with a liquid inlet, a second liquid storage groove, a waste liquid groove, a flow channel and a valve, the liquid storage groove and the waste liquid groove are located at the bottom of the upper shell, the flow channel is used for connecting the second liquid storage groove and the waste liquid groove, and the valve is located at the top of the upper shell; the valve is used for controlling whether the flow channel is connected. The packaging substrate does not contain a high-density integrated circuit; one side of the packaging substrate arranged with the nanopore sequencing chip is provided with a substrate pad corresponding to the first chip pad, and the other side is provided with an arrayed second metal contact; the second metal lead wire is arranged inside the packaging substrate, one end of the second metal lead wire is connected with the substrate pad, and the other end of the second metal lead wire is connected with the second metal contact. The substrate pad is located outside the nanopore sequencing chip, and one end of the second metal lead wire connected with the substrate pad extends to the outside of the packaging substrate and is connected with the first chip pad.

[0081] Preferably, the packaging substrate is a PCB.

[0082] Further, the second plastic sealing shell comprises an upper shell and a bottom shell, the nanopore sequencing chip is located between the upper shell and the bottom shell, and the front surface of the nanopore sequencing chip faces the upper shell.

[0083] Further, the bottom shell is provided with a substrate slot, and the packaging substrate is clamped in the slot.

[0084] Further, the upper shell and the bottom shell are detachably connected.

[0085] Preferably, the packaging structure of the nanopore sequencing chip further comprises a threaded structure, the side surface of the upper shell is provided with a first threaded hole, the side surface of the bottom shell is provided with a second threaded hole corresponding to the first threaded hole, the threaded structure is arranged in the first threaded hole and the second threaded hole, and the upper shell and the bottom shell are fixedly connected through the threaded structure.

[0086] Further, the high polymer gasket is internally provided with a quadrilateral or elliptical second through hole; the second through hole is arranged corresponding to the arrayed holes on the nanopore sequencing chip.

[0087] Further, the bottom shell is arranged on the lower layer, and the bottom shell is attached to the side of the packaging substrate provided with the second metal contact; the side of the packaging substrate provided with the substrate pad is attached to the polymer gasket, and the upper shell covers the surface of the polymer gasket and is fixedly connected to the bottom shell.

[0088] Preferably, the number of substrate pads is selected from one of 120-512; the number of substrate pads, first chip pads and second metal contacts corresponds to the number of holes.

[0089] In the seventh aspect, the application further provides another packaging structure suitable for the nanopore sequencing chip of the first aspect, which can solve the above problems, and differs from the packaging structure of the sixth aspect in that:

[0090] The packaging substrate is provided with an integrated circuit module and a heat conduction module penetrating through the packaging substrate, the integrated circuit module is arranged on the side of the heat conduction module, and the nanopore sequencing chip is arranged on the heat conduction module; the upper and lower surfaces of the packaging substrate are both provided with the integrated circuit module; the side of the packaging substrate is provided with a data transmission interface composed of a metal connection plug, the bottom shell is provided with a third through hole corresponding to the data transmission interface, the data transmission interface can be arranged in the third through hole and extended to the outside of the second plastic package shell.

[0091] In the eighth aspect, the application further provides another packaging structure suitable for the nanopore sequencing chip of the second aspect, which comprises the nanopore sequencing chip of the second aspect, the second plastic package shell, the packaging substrate, the polymer gasket and the second metal lead.

[0092] It differs from the sixth aspect in that:

[0093] The signal transmission assembly is a second signal transmission assembly, which comprises a first electrode line, an integrated circuit area, a third electrode line and a second chip pad connected in sequence, the second chip pad is arranged on the side of the nanopore sequencing chip, the third electrode line is arranged inside the nanopore sequencing chip, and the second chip pad is connected to the integrated circuit area through the third electrode line. The second chip pad is connected to the substrate pad through the second metal lead. The number of second metal contacts, second chip pads and packaging frame pads is correspondingly arranged.

[0094] Further, the packaging structure can directly convert the data measured in real time through the integrated circuit area and output through the second chip pad, which can reduce the interference generated during signal transmission; at the same time, the number of second chip pads required can be much less than the number of holes, thereby improving the density and number of holes on a single chip and the sequencing throughput, further reducing the volume and sequencing cost.

[0095] In summary, the metal lead and the square flat leadless package form in the present application are suitable for nanopore sequencing chips with various numbers of nanopores, can reduce the volume and cost, have good sealing performance, and reduce the crosstalk problem caused by the metal leads; and the nanopore sequencing chip can be transported separately, and the nanopore sequencing chip does not contact any liquid during the transportation, so that the shelf life of the nanopore sequencing chip can be effectively improved.

[0096] The packaging structure of the nanopore sequencing chip can be matched with a clamp to realize manual, semi-automatic and full-automatic film laying.

[0097] Further, the nanopore sequencing chip of the present application can be laid and used immediately and experimental data can be obtained within 30 minutes in combination with the above packaging method and film laying method.

[0098] The present application has the following technical effects:

[0099] The nanopore sequencing chip of the present application has low cost, few processing steps and high yield; the sequencing chip structure can make the wettability of the nanopore good and the conduction rate high by forming a hydrophilic modification area at the bottom of the hole; and the hydrophobicity of the surface of the second insulating layer can be improved by arranging a hydrophobic area on the second insulating layer, so that the generation rate of the oily film and the stability of the oily film are improved. BRIEF DESCRIPTION OF DRAWINGS

[0100] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0101] Figure 1 The nanopore sequencing chip single hole structure schematic diagram provided for embodiment 1 of the present application.

[0102] Figure 2 The nanopore sequencing chip single hole structure front view schematic diagram provided for embodiment 1 of the present application.

[0103] Figure 3 The A-A sectional view of Figure 2 .

[0104] Figure 4 The nanopore sequencing chip single hole actual structure schematic diagram provided for embodiment 1 of the present application.

[0105] Figure 5 The hydrophilic modification schematic diagram provided for embodiment 1 of the present application.

[0106] Figure 6This is a schematic diagram of the hydrophilic modification solution for removing the pore surface provided in Embodiment 1 of the present invention.

[0107] Figure 7 This is a schematic diagram of the packaging structure of the nanopore sequencing chip provided in Embodiment 1 of the present invention.

[0108] Figure 8 This is a schematic diagram of the back structure of the nanopore sequencing chip provided in Embodiment 1 of the present invention.

[0109] Figure 9 This is a schematic diagram of the front view of the unpacked nanopore sequencing chip provided in Embodiment 1 of the present invention.

[0110] Figure 10 This is a cross-sectional view of the packaging structure of the nanopore sequencing chip provided in Embodiment 1 of the present invention.

[0111] Figure 11 This is a cross-sectional view of the liquid storage tank structure provided in Embodiment 1 of the present invention.

[0112] Figure 12 This is a schematic diagram of the ion solution conduction of the nanopore sequencing chip provided in Embodiment 1 of the present invention.

[0113] Figure 13 This is a schematic diagram of the nanopore sequencing chip membrane laying method provided in Embodiment 1 of the present invention.

[0114] Figure 14 This is a schematic diagram of a single pore structure of the nanopore sequencing chip provided in Embodiment 2 of the present invention.

[0115] Figure 15 This is a front view of a single pore structure of the nanopore sequencing chip provided in Embodiment 2 of the present invention.

[0116] Figure 16 for Figure 15 AA cross-section view.

[0117] Figure 17 This is a schematic diagram of the physical structure of a single pore in the nanopore sequencing chip provided in Embodiment 2 of the present invention.

[0118] Figure 18 This is a schematic diagram of the single-pore physical structure of another nanopore sequencing chip provided in Embodiment 2 of the present invention.

[0119] Figure 19 This is a schematic diagram of a single hole structure of the hole sequence provided in Embodiment 3 of the present invention.

[0120] Figure 20 This is a cross-sectional view of a single hole structure in the hole sequence provided in Embodiment 3 of the present invention.

[0121] Figure 21A schematic diagram of the hydrophobic modification of the nanopore sequencing chip provided in Embodiment 3 of the present application.

[0122] Figure 22 An exploded view of the packaging structure of the nanopore sequencing chip provided in Embodiment 3 of the present application.

[0123] Figure 23 A schematic diagram of the packaging structure of the nanopore sequencing chip provided in Embodiment 3 of the present application.

[0124] Figure 24 A schematic diagram of the bottom structure of the upper shell provided in Embodiment 3 of the present application.

[0125] Figure 25 A schematic diagram of the front surface structure of the packaging substrate provided in Embodiment 3 of the present application.

[0126] Figure 26 A schematic diagram of the back surface structure of the packaging substrate provided in Embodiment 3 of the present application.

[0127] Figure 27 A physical diagram of the packaging structure of the nanopore sequencing chip provided in Embodiment 3 of the present application.

[0128] Figure 28 A schematic diagram of the hydrophobic modification provided in Embodiment 4 of the present application.

[0129] Figure 29 A schematic diagram of the packaging substrate structure provided in Embodiment 4 of the present application.

[0130] Figure 30 A schematic diagram of the overall structure of the nanopore sequencing chip provided in Embodiment 5 of the present application.

[0131] Figure 31 A top view of the nanopore sequencing chip provided in Embodiment 5 of the present application.

[0132] Figure 32 A sectional view of the nanopore sequencing chip provided in Embodiment 5 of the present application.

[0133] Figure 33 A further sectional view of the nanopore sequencing chip provided in Embodiment 5 of the present application.

[0134] Figure 34 A schematic diagram of the overall structure of the nanopore sequencing chip provided in Embodiment 7 of the present application.

[0135] Figure 35 A sectional view of the nanopore sequencing chip provided in Embodiment 7 of the present application.

[0136] Figure 36 A schematic diagram of the signal transmission principle of the nanopore sequencing chip provided in Embodiment 5 of the present application.

[0137] Figure 37 A schematic diagram of the packaging structure of the nanopore sequencing chip provided for Example 8 of the present application.

[0138] Figure 38 A water droplet state diagram of the pore surface provided for Comparative Example 1 of the present application.

[0139] Figure 39 A water droplet state diagram of the pore surface provided for Comparative Example 2 of the present application.

[0140] Figure 40 A water droplet state diagram of the pore surface provided for Example 3 of the present application.

[0141] Figure 41 A schematic diagram of the conduction effect of the nanopore sequencing chip provided for Example 1 of the present application.

[0142] Figure 42 A schematic diagram of the film forming effect of the nanopore sequencing chip provided for Example 1 of the present application.

[0143] Figure 43 A schematic diagram of the sequencing effect of the nanopore sequencing chip provided for Example 1 of the present application.

[0144] Explanation of the symbols in the figures:

[0145] 1 - first insulating layer; 101 - monocrystalline silicon layer; 102 - glass layer; 103 - insulating layer; 2 - second insulating layer; 201 - pore; 202 - hydrophilic modification region; 3 - hydrophobic modification solution; 4 - electrode; 5 - hydrophilic modification solution; 6 - boss structure; 7 - groove structure; 8 - packaging frame; 9 - nanopore sequencing chip; 901 - pore sequencing region; 902 - first chip pad; 903 - second chip pad; 904 - third electrode line; 10 - plastic packaging structure; 11 - packaging frame pad; 12 - first metal lead; 13 - first metal contact; 14 - first plastic packaging shell; 15 - first liquid storage tank; 16 - pit structure; 17 - rectangular tooth; 18 - oily film; 19 - ionic solution; 20 - cavity; 21 - upper shell; 211 - liquid inlet; 212 - second liquid storage tank; 213 - waste liquid tank; 214 - flow channel; 215 - valve; 216 - first threaded hole; 22 - bottom shell; 221 - base clamping groove; 222 - second threaded hole; 23 - packaging base; 24 - polymer gasket; 25 - second metal contact; 26 - base pad; 27 - second through hole; 28 - air inlet; 29 - air outlet; 30 - uniform glue tray; 31 - fluorine-containing compound; 32 - heating table; 33 - integrated circuit module; 34 - heat conduction module; 35 - integrated circuit region; 3501 - signal amplifier; 3502 - multiplexer; 3503 - analog-to-digital converter; 36 - first electrode line; 37 - data transmission interface. DETAILED DESCRIPTION

[0146] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only some 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 efforts belong to the scope of protection of the present application.

[0147] It should be understood that, when used in the specification and the appended claims, the terms "comprise" and "include" indicate the presence of the described features, integers, steps, operations, elements, and / or components, but do not exclude one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0148] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", and the like indicate the orientation or positional relationship shown in the drawings, or the orientation or positional relationship commonly used when the product of the present application is used, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0149] In the description of the present application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "arrange", "mount", "connect", and "connect" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be directly connected, or indirectly connected through an intermediate medium, or it can be connected inside 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.

[0150] In the embodiments of the present application, the specific steps for removing the hydrophilic modification solution on the surface of the double-layer-like insulation layer hole structure and retaining the hydrophilic modification solution at the bottom of the hole are as follows:

[0151] The double-layer-like insulation layer structure is cleaned with pure water on its surface and dried, placed on the surface of the tray of the glue spreading machine, the vacuum mode is turned on, the back of the double-layer-like insulation layer hole sequence structure is tightly attached to the surface of the tray, the glue spreading machine drives the double-layer-like insulation layer hole sequence structure to rotate slowly, then the surface of the double-layer-like insulation layer hole sequence structure is washed with a high-pressure water gun until the hydrophilic modification solution on the upper part of the hole and the surface of the second insulation layer is cleaned and the hydrophilic modification solution remains at the bottom of the hole; then the water on the surface of the double-layer-like insulation layer hole sequence structure is blown dry with an air gun, so that the moisture on the surface of the second insulation layer is removed and the hole remains in a wet state; the angle between the nozzle of the high-pressure water gun and the surface thereof is 15-60°; the angle between the nozzle of the air gun and the surface thereof is maintained at 15-60°.

[0152] Example 1:

[0153] Referring to Figures 1-4 The nanopore sequencing chip shown in the figure comprises a first insulating layer 1, a second insulating layer 2, and an electrode 4, the second insulating layer 2 is arranged on the first insulating layer 1, and the second insulating layer 2 is provided with an array of holes 201; the hole 201 penetrates through the second insulating layer 2 and communicates with the first insulating layer 1; the electrode 4 is arranged at the bottom of the hole 201; the bottom of the hole 201 is provided with a hydrophilic modification area 202; the hydrophilic modification area 202 is obtained by modification with a hydrophilic modification solution, and the hydrophilic modification solution is a chitosan solution; the second insulating layer 2 is provided with a hydrophobic area, in this embodiment, the hydrophobic area is an oil storage area, and the oil storage area is arranged around each hole 201.

[0154] Specifically, the upper surface of the second insulating layer 2 is provided with a plurality of hexagonal boss structures 6, and the space between the plurality of hexagonal boss structures 6 forms a regular flow channel, and the regular flow channel and the hexagonal boss structure 6 form a honeycomb type oil storage area. The regular flow channel can guide the reflux of the ion solution 19 into the hole 201, so that a stable water-in-oil structure is formed in the hole 201. The second insulating layer 2 and the honeycomb type oil storage area can form a double-layer insulating layer hole sequence structure.

[0155] In this embodiment, the plurality of hexagonal boss structures 6 can support the oily film on the hole surface, and the honeycomb type oil storage area can improve the hydrophobicity of the surface of the second insulating layer 2, which is conducive to the drainage and storage of the organic solution, thereby facilitating the formation and fixation of the oily film 18.

[0156] Specifically, the distance between the hexagonal boss structure 6 and the edge of the hole 201 is 3 μm; the height of the hole 201 is 40 μm; and the height of the oil storage area is 3 μm.

[0157] Specifically, in this embodiment, the size of the electrode 4 is greater than the pore size of the hole 201, so as to ensure the contact area of the electrode 4 and the ion solution 19.

[0158] Specifically, referring to Figure 9 , the nanopore sequencing chip 9 further comprises a first signal transmission component, the first signal transmission component comprises a second electrode line (not shown in the figure) corresponding to the number of holes 201 and a first chip pad 902, the first chip pad 902 is arranged on the side of the nanopore sequencing chip 9, the second electrode line is arranged in the interior of the nanopore sequencing chip 9, and the first chip pad 902 is connected with the electrode 4 through the second electrode line.

[0159] Specifically, the preparation method of the nanopore sequencing chip of this embodiment comprises the following steps:

[0160] S1, disposing an electrode 4 on the first insulating layer 1, then coating photoresist on the surface of the first insulating layer 1, preparing the second insulating layer 2 on the first insulating layer 1 according to the diffraction characteristics of light by photoetching, and forming holes 201 corresponding to the electrode 4 on the second insulating layer 2, at the same time forming hexagonal prismatic bosses around the top of the holes 201, and further forming a honeycomb type oil storage area on the second insulating layer 2, so that a double-layer insulating layer hole sequence structure can be formed by one photoetching;

[0161] S2, hydrophilic modification; as shown in the figure, the double-layer insulating layer hole sequence structure is soaked in a hydrophilic modification solution 5 with a mass concentration of 5%, so that the hydrophilic modification solution 5 infiltrates the holes 201, and the hydrophilic modification solution layer is obtained on the surface of the double-layer insulating layer hole sequence structure under the condition of temperature 30℃ for 60min. Figure 5

[0162] S3, as shown in the figure, part of the hydrophilic modification solution 5 on the surface of the double-layer insulating layer structure is removed and the hydrophilic modification solution 5 at the bottom of the hole 201 is reserved, so that the upper surface of the double-layer insulating layer structure and the mouth of the hole 201 return to a hydrophobic state; aging for 6 days, so that the hydrophilic modification area 202 is formed at the bottom of the hole 201 under the action of the hydrophilic modification solution 5, and finally cleaned with a high-pressure water gun to remove the crystalline substance, blow dry and heat dry the moisture to obtain a nanopore sequencing chip. Figure 6

[0163] Specifically, in this embodiment, the specific steps of removing part of the hydrophilic modification solution 5 on the surface of the double-layer insulating layer structure and reserving the hydrophilic modification solution 5 at the bottom of the hole in step S3 are as follows:

[0164] The double-layer insulating layer structure is cleaned with pure water and dried, placed on the surface of the tray of the spin coater, and the opposite sides of the double-layer insulating layer structure are tightly attached to the surface of the tray. The spin coater drives the double-layer insulating layer structure to rotate at a speed of 100r / s, and then the surface is washed with a high-pressure water gun until only the hydrophilic modification solution 5 remains at the bottom of the hole 201; then the water on the surface is blown dry with an air gun, so that the moisture on the surface of the second insulating layer 2 is removed and the hole 201 remains wet; the angle between the nozzle of the high-pressure water gun and the surface is 15°; the angle between the nozzle of the air gun and the surface is maintained at 60°.

[0165] Specifically, in this embodiment, the hydrophilic modification solution 5 is a chitosan solution, and the solvent of the chitosan solution is an acetic acid solution prepared by diluting 1mol / L acetic acid with pure water by 100 times;

[0166] ​​Specifically, the surface of the nanopore sequencing chip prepared by the embodiment has a smaller size of the honeycomb-like structure, which can effectively improve the hydrophobicity of the surface of the second insulating layer 2, and further improve the stability of the thin film after film formation through the oil storage area. Secondly, the nanopore sequencing chip of the embodiment can obtain a double-layer insulating layer pore sequence structure through one-time photolithography exposure, which is simple and reliable in process, short in processing time and low in cost. It also does not require high-resolution mask plates and high-end photolithography machines, and can be processed using ordinary exposure methods and ordinary photoresists of ordinary photolithography machines.

[0167] For further reference Figures 7-11 The embodiment also provides a packaging structure of a nanopore sequencing chip, which comprises a plastic packaging structure 10 and the nanopore sequencing chip 9 described above. The plastic packaging structure 10 comprises a first plastic packaging shell 14, a packaging frame 8 and a first metal lead 12. The first plastic packaging shell 14 is provided with a first through hole, and the packaging frame 8 is mounted at the bottom of the first through hole. The packaging frame 8 is used for bearing the nanopore sequencing chip 9 and dissipating heat to achieve the effect of temperature control. The nanopore sequencing chip 9 is clamped in the packaging frame 8. A first liquid storage groove 15 is formed between the upper end of the first through hole and the upper surface of the nanopore sequencing chip 9.

[0168] The first metal lead 12 is located above the nanopore sequencing chip 9. Each first chip pad 902 and packaging frame pad 11 is connected through the first metal lead 12. The first plastic packaging shell 14 is provided with a first metal contact 13 outside the bottom for connecting conduction. The pores 201 on the nanopore sequencing chip 9 form a pore sequencing area 901, which is located in the middle of the nanopore sequencing chip 9, so that the pore sequencing area 901 is completely exposed in the first liquid storage groove 15 to further ensure the film formation rate of the nanopore sequencing chip 9.

[0169] Specifically, in the embodiment, the pores 201, the first chip pads 902, the packaging frame pads 11 and the first metal contacts 13 are all provided with 96. The packaging frame 8 adopts a metal frame.

[0170] Specifically, in the embodiment, the metal lead and the square flat leadless packaging form are adopted for packaging, which can reduce the cost, has good sealing performance and reduces the crosstalk problem caused by the metal leads. In addition, the nanopore sequencing chip does not integrate functional modules such as signal processing circuits. The nanopore sequencing chip is easy to clean and has high reusability, which reduces the sequencing cost of users.

[0171] Specifically, the film laying method of the nanopore sequencing chip in the embodiment comprises the following steps:

[0172] (1) A pipette is used to drop ion solution 19 on the surface of the nanopore sequencing chip 9, so that the ion solution 19 completely fills the liquid storage groove 15 of the plastic packaging structure 10. At this time, the ion solution 19 cannot infiltrate into the pores 201;

[0173] (2) repeatedly draw and release liquid on the surface of the nanopore sequencing chip 9 until the ion solution 19 enters the hole 201, so that the hole 201 is conducted, as shown in Figure 12 ;

[0174] (3) use a pipette to draw an organic solution and bubble on the surface of the hole 201, as shown in Figure 13 , the organic solution is fixed into an oily film 18 under the hydrophobic and oleophilic effect of the surface structure of the nanopore sequencing chip structure, so that the ion solution 19 in the hole 201 is isolated from the ion solution 19 outside the hole 201.

[0175] Specifically, the nanopore sequencing chip film laying method of the embodiment is simple, and can quickly conduct, lay film, and embed protein holes and other operations. Data can be obtained within 30 minutes; the instant film laying method has a long service life;

[0176] Example 2

[0177] The difference between this embodiment and Example 1 is the structure of the nanopore sequencing chip, the hydrophilic modification solution, the preparation method and the packaging structure. The specific differences are as follows:

[0178] Please refer to the nanopore sequencing chip shown in Figures 14-18 , in this embodiment, the oil storage area is composed of a plurality of groove structures 7 arranged on the second insulating layer 2, and the second insulating layer 2 and the groove structure 7 form a double-layer insulating layer hole sequence structure; the inner side along the edge of the top of the hole 201 is provided with a pit structure 16; the distance between the groove structure 7 and the edge of the hole 201 is 10 μm; the height of the hole 201 is 50 μm; the height of the oil storage area is 5 μm. The groove structure 7 can improve the hydrophobicity of the surface of the second insulating layer 2, support the film on the surface of the hole, and at the same time, the groove structure 7 is beneficial to the storage of the organic solvent, thereby facilitating the formation and fixation of the oily film 18. Specifically, in this embodiment, the hydrophilic modification solution is a dopamine solution.

[0179] Specifically, in this embodiment, the preparation method of the nanopore sequencing chip includes the following steps:

[0180] S1, arrange the electrode 4 on the first insulating layer 1, then coat photoresist on the first insulating layer 1, prepare the second insulating layer 2 on the first insulating layer 1 according to the diffraction characteristics of light, and form the hole 201 corresponding to the electrode 4 on the second insulating layer 2, at the same time, form the groove structure 7 arranged around the hole 201, and then form the groove type oil storage area on the surface of the second insulating layer 2; so that a double-layer insulating layer hole sequence structure can be formed by one-time photoetching exposure;

[0181] S2, hydrophilic modification; the double-like insulating layer hole sequence structure is modified by immersing in a dopamine solution with a mass concentration of 10%, so that the dopamine solution infiltrates the holes 201, and the modification is performed at a temperature of 20 DEG C for 180 min, and a dopamine solution layer is obtained on the surface of the double-like insulating layer hole sequence structure.

[0182] S3, part of the dopamine solution layer on the surface of the double-like insulating layer structure is removed and the dopamine solution layer at the bottom of the holes is retained, so that the upper surface of the hole sequence structure and the nanopore mouth are restored to a hydrophobic state; aging for 6 days, so that the bottom of the hole 201 forms a hydrophilic modification area 202 under the action of the dopamine solution, and a nanopore sequencing chip is obtained.

[0183] Specifically, in this embodiment, in step S3, the specific steps of removing the dopamine solution layer on the surface of the double-like insulating layer structure and retaining the dopamine solution layer at the bottom of the hole are as follows:

[0184] The double-like insulating layer structure is cleaned with pure water and dried, and the opposite surface of the structure is placed on the surface of the tray of the glue spreader, the vacuum mode is turned on, and the opposite surface of the double-like insulating layer structure is tightly attached to the surface of the tray. The hole sequence structure is slowly rotated at a speed of 100 r / s by the glue spreader, and then the surface is washed with a high-pressure water gun until the dopamine solution layer on the upper part of the hole 201 and the upper surface of the second insulating layer is cleaned and the bottom of the hole 201 is left with a dopamine solution layer. Then, the water on the surface is blown dry with an air gun, so that the moisture on the surface of the second insulating layer is removed and the hole 201 remains wet. The angle between the nozzle of the high-pressure water gun and the surface is 30 DEG, and the angle between the nozzle of the air gun and the surface is maintained at 30 DEG.

[0185] Specifically, the groove structure of the nanopore sequencing chip prepared by this embodiment has a smaller size, which can effectively improve the hydrophobicity of the surface of the second insulating layer 2, and further improve the stability of the thin film after film formation through the oil storage area. Secondly, the nanopore sequencing chip of this embodiment can obtain a double-like insulating layer hole sequence structure by one-time photolithography exposure, which is simple and reliable in process, short in processing time and low in cost. It does not need high-resolution mask and high-end photolithography machine, and can be processed by using ordinary exposure mode and ordinary photoresist of ordinary photolithography machine.

[0186] Specifically, the difference between the packaging structure of the nanopore sequencing chip in this embodiment and in embodiment 1 is that the packaging frame 8 is replaced by a silicon substrate.

[0187] Embodiment 3

[0188] The difference between this embodiment and embodiment 1 is the structure of the nanopore sequencing chip, the hydrophilic modification solution, the preparation method and the packaging structure, and the specific differences are as follows:

[0189] In this embodiment, the hydrophobic region is a hydrophobic modified region. The top of the hole 201 is surrounded by a smooth planar structure, the height of the hole 201 is 50 μm, and the diameter is 80 μm; the top of the hole 201 is provided with a hydrophobic modified region, which can achieve a surface hydrophobic effect, thereby enhancing the adhesion of the second insulating layer 2 surface and the oily film 18, facilitating the formation and fixation of the film, and forming a stable water-in-oil structure in the hole 201.

[0190] Specifically, in this embodiment, the hydrophilic modification solution 5 is a polylysine solution.

[0191] Specifically, the preparation method of the nanopore sequencing chip in this embodiment includes the following steps:

[0192] S1, setting an electrode 4 on the first insulating layer 1, then coating photoresist on the upper surface of the first insulating layer 1, and preparing a second insulating layer 2 on the first insulating layer 1 by first photoetching exposure, and forming a hole 201 corresponding to the electrode 4 in communication on the second insulating layer 2, to obtain a single-layer hole sequence structure, as shown in Figure 19 、 Figure 20 ;

[0193] S2, hydrophilic modification; the single-layer hole sequence structure is soaked and modified with a polylysine solution with a mass concentration of 8%, so that the polylysine solution infiltrates the hole 201, and the modification is carried out at a temperature of 50°C for 30 minutes;

[0194] S3, removing part of the polylysine solution on the surface of the structure modified in S2 and retaining the polylysine solution at the bottom of the hole 201, so that the surface returns to a hydrophobic state; aging for 6 days, so that the bottom of the hole 201 forms a hydrophilic modified region 202 under the action of the polylysine solution, to obtain a hydrophilic modification chip;

[0195] S4, hydrophobic modification; the hydrophilic modification chip obtained in step S3 is subjected to hydrophobic modification; a dispensing machine is used to add a hydrophobic modification solution 3 at the center of the surface of the second insulating layer 2, as shown in Figure 21 In this embodiment, the hydrophobic modification solution 3 is a polysilicon compound, and the concentration of the polysilicon compound is 10%-60%. Specifically, in this embodiment, the content of the polysilicon compound is 29%. The volume of the added polysilicon compound is 0.3 μL-5 μL. Specifically, in this embodiment, the volume of the added polysilicon compound is 0.5 μL. The volume of the polysilicon compound needs to be adjusted according to the changes in the diameter, height and number of the holes. If the polysilicon compound exceeds the range, it will easily infiltrate the entire hole 201, so that the hydrophilic modified region 202 at the bottom of the hole 201 and the electrode 4 are covered with the polysilicon compound, resulting in the inability of the hole 201 to conduct during biochemical experiments.

[0196] S5, after the polysilicon compound covers the surface of the second insulating layer 2 and the top of the hole 201, it is heated in a ventilated environment, modified at a temperature of 150℃ for 30min, a hydrophobic modification zone is formed on the top of the hole 201, and a nanopore sequencing chip is obtained.

[0197] Specifically, in the present embodiment, the polylysine solution on the surface of the single-layer hole sequence structure is removed and the polylysine solution at the bottom of the hole is retained, and the specific steps are as follows:

[0198] The hole sequence structure is washed with pure water and dried, and the opposite surface of the structure is placed on the surface of the tray of the glue spreading machine. The vacuum mode is turned on, and the opposite surface of the double-layer insulating layer hole sequence structure is tightly attached to the surface of the tray. The glue spreading machine drives the hole sequence structure to rotate slowly at a speed of 100r / s. Then the surface of the hole sequence structure is washed with a high-pressure water gun until the hydrophilic modification solution on the upper part of the hole and the surface of the second insulating layer is washed clean and the hydrophilic modification solution remains at the bottom of the hole. Then the water on the surface of the hole sequence structure is blown dry with an air gun, so that the water on the surface of the second insulating layer is removed and the hole remains in a wet state. The angle between the nozzle of the high-pressure water gun and the surface of the hole sequence structure is 60°, and the angle between the nozzle of the air gun and the surface of the hole sequence structure is maintained at 15°.

[0199] Specifically, please refer to Figures 22-27 In the present embodiment, the packaging structure of the nanopore sequencing chip comprises the nanopore sequencing chip, a second plastic packaging shell, a packaging substrate 23 and a high polymer gasket 24. The nanopore sequencing chip is arranged on the packaging substrate 23, the packaging substrate 23 is arranged in the second plastic packaging shell, and the high polymer gasket 24 is arranged between the nanopore sequencing chip and the second plastic packaging shell.

[0200] Specifically, the second plastic packaging shell comprises an upper shell 21 and a bottom shell 22, and the nanopore sequencing chip is located between the upper shell 21 and the bottom shell 22, and the front surface of the nanopore sequencing chip faces the upper shell 21.

[0201] Specifically, the upper shell 21 is provided with a liquid inlet 211, a second liquid storage groove 212, a waste liquid groove 213, a flow channel 214 and a valve 215. The second liquid storage groove 212 and the waste liquid groove 213 are located at the bottom of the upper shell 21. The flow channel 214 is used to connect the second liquid storage groove 212 and the waste liquid groove 213. The valve 215 is located at the top of the upper shell 21, and the valve 215 is used to control whether the flow channel 214 is connected. The bottom shell 22 is provided with a substrate clamping groove 221, and the packaging substrate 23 is clamped in the substrate clamping groove 221.

[0202] Specifically, it also comprises a threaded structure, the side surface of the upper shell 21 is provided with a first threaded hole 216, the side surface of the bottom shell 22 is provided with a second threaded hole 222 corresponding to the first threaded hole 216, the threaded structure is arranged in the first threaded hole 216 and the second threaded hole 222, and the upper shell 21 and the bottom shell 22 are fixedly connected through the threaded structure.

[0203] Specifically, in the embodiment, the packaging substrate 23 does not contain an integrated circuit.

[0204] Specifically, in the embodiment, the nanopore sequencing chip 9 is provided with a first signal transmission assembly, the first signal transmission assembly includes a first chip pad 902 and a second electrode wire, the first chip pad 902 is arranged on the side of the nanopore sequencing chip 9, and the second electrode wire is arranged in the interior of the nanopore sequencing chip 9 and connected with the first chip pad 902 and the electrode 4. The nanopore sequencing chip 9 is arranged on the packaging substrate 23.

[0205] Specifically, the packaging substrate 23 is provided with a substrate pad 26 on one side of the nanopore sequencing chip 9 and is provided with an array of second metal contacts 25 on the other side, a second metal lead wire is arranged in the interior of the packaging substrate 23, one end of the second metal lead wire is connected with the substrate pad 26, and the other end of the second metal lead wire is connected with the second metal contacts 25. The substrate pad 26 is arranged on the side of the nanopore sequencing chip 9 and corresponds to the first chip pad 902; one end of the second metal lead wire connected with the substrate pad 26 extends to the outside of the packaging substrate 23 and is connected with the first chip pad 902.

[0206] Specifically, the high polymer gasket 24 is internally provided with an oval-shaped second through hole 27, and the second through hole 27 corresponds to the nanopore sequencing region 901 on the nanopore sequencing chip 9.

[0207] Specifically, the bottom shell 22 is arranged on the lower layer, the bottom shell 22 is attached to the side of the packaging substrate 23 provided with the second metal contacts 25, the side of the packaging substrate 23 provided with the substrate pad 26 is attached to the high polymer gasket 24, and the upper shell 21 covers the surface of the high polymer gasket 24 and is fixedly connected with the bottom shell 22.

[0208] Specifically, in the embodiment, the packaging substrate 23 does not include an integrated signal processing circuit and other functional modules, but only contains a simple connection circuit.

[0209] Specifically, in the embodiment, the nanopore 201, the substrate pad 26, the second metal contact 25 and the first chip pad 902 are each provided with 512.

[0210] Embodiment 4

[0211] The embodiment is different from the embodiment 1 in the structure, preparation method and packaging structure of the nanopore sequencing chip, and the specific differences are as follows:

[0212] In the embodiment, the height of the oil storage region is 20 μm, the hydrophobic region further includes a hydrophobic modified region, and the hydrophobic modified region is arranged on the top of the nanopore 201.

[0213] In this embodiment, the preparation method of the nanopore sequencing chip is different from that of embodiment 1 in that after the hydrophilic modification region is formed in step S3, a hydrophobic modification is further performed on the top of the hole 201.

[0214] Specifically, as shown in Figure 28 , the hydrophobic modification in this embodiment is performed in a closed transparent cavity 20, the top of the cavity 20 is provided with an air inlet 28 and an air outlet 29, the bottom center is provided with a glue uniformizing tray 30, and the two sides are provided with heating tables 32.

[0215] Specifically, the hydrophobic modification in this embodiment includes the following steps: placing the chip that has been hydrophilically modified in the center of the glue uniformizing tray 30, and dropping fluorine-containing compound 31 on the heating table 32; the content of the fluorine-containing compound 31 is 10%; the volume of the fluorine-containing compound 31 dropped is 5 μL. Heat at a temperature of 100°C for 1 min, and then stop heating; during this period, the glue uniformizing tray 30 rotates at a speed of 100 r / s with the hydrophilically modified chip and cools down, and high-purity nitrogen gas is continuously input at the air inlet; the continuously supplied nitrogen gas can exhaust a large amount of fluorine-containing gas and make the fluorine-containing gas cool down and uniformly deposit on the surface of the second insulating layer 2 to form a hydrophobic modification region; if the content of the modification reagent is not diluted or nitrogen is not introduced, the deposited hydrophobic film will be too thick, which will cause the subsequent biological experiment to be unable to conduct or the performance to be uneven.

[0216] Specifically, the difference between the packaging structure of the nanopore sequencing chip in this embodiment and that of embodiment 3 is that the packaging substrate 23 of this embodiment is provided with an integrated circuit module 33 and a heat conduction module 34 penetrating through the packaging substrate 23, as shown in Figure 29 , the integrated circuit module 33 is arranged on the upper and lower surfaces of the packaging substrate 23, the side surface of the packaging substrate 23 is provided with a data transmission interface 37 composed of metal connecting plugs, and the signals in the integrated circuit module 33 are transmitted through the data transmission interface 37. The bottom shell 22 is provided with a third through hole corresponding to the data transmission interface 37, the data transmission interface 37 can be arranged in the third through hole, and extends to the outside of the second plastic packaging shell.

[0217] Specifically, the hole 201, the substrate pad 26, the second metal contact 25 and the first chip pad 902 in this embodiment are each provided with 384.

[0218] Embodiment 5

[0219] For details, please further refer to Figures 30-33 , the difference between this embodiment and embodiment 1 is that:

[0220] In the nanopore sequencing chip of the embodiment, the first signal transmission assembly is replaced by a second signal transmission assembly, the second signal transmission assembly comprises a third electrode line 904, an integrated circuit area 35, a first electrode line 36 and a second chip pad 903 connected in sequence, the second chip pad 903 is arranged on the side of the nanopore sequencing chip 9, the third electrode line 904 is arranged inside the nanopore sequencing chip 9, and the second chip pad 903 is connected with the integrated circuit area 35 through the third electrode line 904.

[0221] Specifically, in the embodiment, the integrated circuit area 35 comprises a signal amplifier 3501, a multiplexer 3502 and an analog-to-digital converter 3503 connected in sequence through a fourth electrode line.

[0222] Specifically, the first insulating layer 1 comprises a substrate layer and an insulating layer 103, the insulating layer 103 is arranged between the second insulating layer 2 and the substrate layer, and the integrated circuit area 35 is arranged inside the substrate layer.

[0223] Specifically, the electrode 4 is arranged on the substrate layer and communicates with the outside of the first insulating layer 1, the first electrode line 36 is arranged inside the substrate layer and connects the electrode 4 and the integrated circuit area 35.

[0224] Specifically, the holes 201 arranged in an array on the second insulating layer 2 form a pore sequencing area 901 below the integrated circuit area 35.

[0225] Specifically, the substrate layer in the embodiment is a single crystal silicon layer 101, and the integrated circuit area 35 is arranged inside the single crystal silicon layer 101.

[0226] Specifically, the second chip pad 903 is connected through the first metal lead 12 and the packaging frame pad 11.

[0227] Specifically, please further refer to Figure 33 After the electrode 4 receives the signal, the current signal can be transmitted to the signal amplifier 3501 through the first electrode line 36, the current signal is transmitted to the multiplexer 3502 through the fourth electrode line after passing through the signal amplifier 3501, the current signal of multiple electrodes 4 is received by one multiplexer 3502 at the same time, the current signal is transmitted to the analog-to-digital converter 3503 through the fourth electrode line, the analog-to-digital converter 3503 can convert the current signal into a digital signal, and then the digital signal is transmitted to the second chip pad 903 through the third electrode line 904 and output through the second chip pad 903.

[0228] Specifically, the holes 201 in the embodiment are provided in 1920, and the second chip pad 903, the packaging frame pad 11 and the first metal contact 13 are each provided in 96.

[0229] In the embodiment, the number of the second chip pads 903, the packaging frame pads 11 and the first metal contacts 13 does not need to correspond to the number of the holes 201 one by one, since the multiplexer 3503 can receive the current signals of multiple electrodes 4 at the same time, the number of the holes 201 can be set as an integer multiple of 96, the current signals can be converted by the integrated circuit area 35 directly to output the real-time measured data, which can reduce the interference generated during signal transmission, increase the number of the holes 201 and the flux, and further reduce the volume.

[0230] Embodiment 6

[0231] The difference between the embodiment and embodiment 5 is that the number of the holes 201 in the embodiment is 1536.

[0232] Embodiment 7

[0233] The difference between the embodiment and embodiment 3 is the nanopore sequencing chip and the packaging structure.

[0234] For details, please refer to Figure 34 and Figure 35 The difference between the nanopore sequencing chip of the embodiment and that of embodiment 3 is that:

[0235] The nanopore sequencing chip 9 of the embodiment further comprises an integrated circuit area 35 and a first electrode line 36, the integrated circuit area 35 is arranged on the first insulating layer 1 and connected to the electrodes 4 through the first electrode line 36.

[0236] Specifically, the first insulating layer 1 comprises a substrate layer and an insulating layer 103, the insulating layer 103 is arranged between the second insulating layer 2 and the substrate layer; the integrated circuit area 35 is located between the substrate layer and the insulating layer 103.

[0237] Specifically, the electrodes 4 are arranged on the substrate layer and communicate with the outside of the first insulating layer 1, the first electrode line 36 is arranged in the inside of the substrate layer and connects the electrodes 4 and the integrated circuit area 35.

[0238] Specifically, the holes 201 arranged in an array on the second insulating layer 2 form a hole sequencing area 901, and the integrated circuit area 35 is arranged on the side of the hole sequencing area 901.

[0239] Specifically, the substrate layer in the embodiment comprises a single crystal silicon layer 101 and a glass layer 102, the integrated circuit area 35 is arranged between the single crystal silicon layer 101 and the insulating layer 103, and the single crystal silicon layer 101 is located between the glass layer 102 and the second insulating layer 2.

[0240] The difference between the packaging structure of the nanopore sequencing chip of the embodiment and that of embodiment 3 is that the packaging substrate in the embodiment does not need to be provided with an integrated circuit module and a large number of substrate pads 26.

[0241] Specifically, the holes 201 are provided with 1000, and the second chip pads 903, the substrate pads 26 and the second metal contacts 25 are each provided with 10.

[0242] In the embodiment, the number of the second chip pads 903, the substrate pads 26 and the second metal contacts 25 does not need to correspond to the number of the holes 201, and the real-time measured data can be converted directly through the integrated circuit area 35 and output through the second chip pads 903, which can reduce the interference generated during signal transmission, increase the number and flux of the holes 201, and further reduce the volume.

[0243] Embodiment 8

[0244] The difference between the embodiment and embodiment 1 is only that the window structure and the size of the first plastic package shell are different in the embodiment.

[0245] The first liquid storage tank 15 is composed of two groups of opposite side walls, the lower side wall in the group of opposite side walls with a shorter side length is a vertical structure, and the upper side wall is a chamfered design, and the other group of opposite side walls is a vertical structure; the chamfered design is helpful for fluid diffusion and buffering, and improves the performance of the chip.

[0246] In order to illustrate the technical effects of the present application, the nanopore sequencing chip prepared in embodiment 1 is subjected to manual hole conduction experiment, film coating experiment and biological test experiment. The test results are shown in Table 1. Figures 41-43 As shown in Table 1, the hole wetting rate of embodiment 1 is as high as 97% or more, the film coating rate and film forming rate are high, and the use is convenient, the test efficiency is high, and the data can be obtained within 30 minutes.

[0247] In order to further illustrate the technical effects of the present application, comparative example 1 and comparative example 2 are set based on embodiment 3.

[0248] Among them, the difference between comparative example 1 and embodiment 3 is only that the nanopore sequencing chip is not subjected to hydrophilic modification or hydrophobic modification; the difference between comparative example 2 and embodiment 3 is only that the hydrophilic modification solution is not removed during hydrophilic modification.

[0249] Drop water test is carried out on embodiment 3, comparative example 1 and comparative example 2, that is, pure water is added dropwise on the top of the holes of the nanopore sequencing chips of comparative example 1, comparative example 2 and embodiment 3, and the water drop state is observed. The test results are shown in Table 2. Figures 38-40 As shown in Table 2, after the nanopore sequencing chip treated by the method of the present application is added with pure water dropwise on the top of the holes, the water drop state is more round compared with comparative example 1 and comparative example 2, which shows that the treatment scheme of the present application can obviously improve the hydrophobicity of the top of the holes.

[0250] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any skilled person in the art can easily think of various equivalent modifications or replacements within the technical range disclosed by the present application, and these modifications or replacements shall be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. A nanopore sequencing chip, characterized in that, The application relates to a microfluidic chip, which comprises a first insulating layer, a second insulating layer and an electrode, wherein the second insulating layer is arranged on the first insulating layer, a plurality of holes are arranged on the second insulating layer in an array, the holes penetrate through the second insulating layer and are communicated with the first insulating layer, the electrode is arranged at the bottom of the hole, a hydrophobic area is arranged on the second insulating layer and is located above the hole and surrounds the hole, a hydrophilic modification area is arranged at the bottom of the hole, the hydrophilic modification area is obtained by modification of a hydrophilic modification solution, and the hydrophilic modification solution is selected from one of a chitosan solution, a dopamine solution and a polylysine solution. The hydrophobic area comprises an oil storage area, the oil storage area is arranged on the upper surface of the second insulating layer, a plurality of groove structures or a plurality of convex structures are arranged on the upper surface of the second insulating layer, the space between the plurality of convex structures or the plurality of groove structures forms the oil storage area, and the distance between the oil storage area and the hole edge is 3 mu m to 50 mu m.

2. The nanopore sequencing chip of claim 1, wherein, The convex structure is circular, quasi-circular or polygonal, and the polygonal structure comprises a regular polygon and an irregular polygon.

3. The nanopore sequencing chip of claim 2, wherein, The convex structure is hexagonal, a plurality of hexagonal convex structures are regularly arranged on the second insulating layer, and the distance between adjacent hexagonal convex structures is 1 mu m to 50 mu m.

4. The nanopore sequencing chip of claim 3, wherein, The plurality of hexagonal convex structures form regular flow channels, the regular flow channels and the hexagonal convex structures form a honeycomb structure, and the depth of the regular flow channel is 1 mu m to 50 mu m.

5. The nanopore sequencing chip of claim 1, wherein, The upper surface of the second insulating layer is provided with a plurality of groove structures, the plurality of groove structures form the oil storage area, the groove structure is circular or polygonal, the plurality of groove structures are regularly arranged to form the oil storage area, and the depth of the groove structure is 1 mu m to 50 mu m.

6. The nanopore sequencing chip of claim 1, wherein, The hydrophobic area further comprises a hydrophobic modification area, the hydrophobic modification area is obtained by hydrophobic modification of a hydrophobic modification solution, and the hydrophobic modification solution is selected from one of a fluorine-containing compound, a polysilicon compound and a synthetic carbon chain compound.

7. The nanopore sequencing chip of claim 6, wherein, The mass concentration of the hydrophobic modification solution is 10% to 60%, and the hydrophobic modification comprises the following steps. The hydrophobic modification solution is dropped on the surface of the second insulating layer, so that the hydrophobic modification solution covers the upper surface of the second insulating layer and the upper end of the hole without penetrating into the bottom of the hole; then the hydrophobic modification solution is heated at a temperature of 100 DEG C to 200 DEG C for 30 min to 180 min to form the hydrophobic area above the hole; The volume of the hydrophobic modification solution is 0.5 mu L to 5 mu L.

8. The nanopore sequencing chip of claim 7, wherein, The hydrophobic modification solution is selected from a fluorine-containing compound, and the hydrophobic modification comprises the following steps. The fluorine-containing compound is heated to evaporate the fluorine-containing compound into a gaseous state, high-purity nitrogen or inert gas is used to drive the gaseous fluorine-containing compound to fill the modification environment and uniformly deposit on the surface of the second insulating layer to form the hydrophobic area.

9. The nanopore sequencing chip of any one of claims 1-8, wherein, The microfluidic chip further comprises a signal transmission assembly, the signal transmission assembly is connected with the electrode and is used for transmitting a test signal.

10. The nanopore sequencing chip of claim 9, wherein, The signal transmission assembly comprises an electrode line and a chip pad, the chip pad is arranged outside the first insulating layer or the second insulating layer, and the chip pad is connected with the electrode through the electrode line.

11. The nanopore sequencing chip of claim 10, wherein, The signal transmission component further comprises an integrated circuit region and a first electrode line, the integrated circuit region is arranged on the first insulating layer and connected with the electrode through the first electrode line, one end of the electrode line is connected with a chip pad, and the other end is connected with the integrated circuit region and connected with the electrode through the integrated circuit region and the first electrode line; the integrated circuit region comprises a signal amplifier, a multiplexer and an analog-digital converter; the signal amplifier, the multiplexer and the analog-digital converter are sequentially connected through a fourth electrode line.

12. The nanopore sequencing chip of claim 11, wherein, The first insulating layer comprises a base layer and an insulating layer, the insulating layer is arranged between the second insulating layer and the base layer; the integrated circuit region is located between the base layer and the insulating layer or arranged inside the base layer. The electrode is arranged on the upper surface of the base layer, and the upper surface of the electrode is in communication with the outside of the first insulating layer; the first electrode line is arranged inside the base layer and connected with the electrode and the integrated circuit region.

13. The nanopore sequencing chip of claim 12, wherein, The base layer comprises a monocrystalline silicon layer, and the integrated circuit region is arranged on the monocrystalline silicon layer.

14. The nanopore sequencing chip of claim 13, wherein, The base layer further comprises a glass layer, and the monocrystalline silicon layer is located between the glass layer and the insulating layer.

15. The nanopore sequencing chip of claim 11, wherein, The integrated circuit region is provided with a plurality of.

16. The nanopore sequencing chip of any one of claims 10-15, wherein, The preparation method of the hydrophilic modification region comprises the following steps: S1, hydrophilic modification; the pore is soaked with a hydrophilic modification solution with a mass concentration of 5%-20% and modified for 30-180 min at a temperature of 20-50 ℃; S2, the hydrophilic modification solution at the pore opening part is removed and the hydrophilic modification solution at the pore bottom part is retained, so that the upper end of the pore returns to a hydrophobic state; and then the pore is aged for more than 5 days, so that the pore bottom part forms a hydrophilic modification region under the action of the hydrophilic modification solution.

17. The nanopore sequencing chip of claim 16, wherein, The pore is completely soaked into the hydrophilic modification solution, the surface direction of the second insulating layer needs to be consistent with the normal direction of the liquid surface, and the bubbles inside the pore need to be completely discharged, so that the inner wall of the pore is completely soaked with the hydrophilic modification solution.

18. A method of fabricating a nanopore sequencing chip as claimed in any one of claims 1 to 8, characterised by, The method comprises the following steps: An electrode is arranged on the first insulating layer, photoresist is coated on the surface of the first insulating layer to form a photoresist surface, a second insulating layer is prepared on the first insulating layer by a photoetching technology according to the diffraction characteristics of light, so that the pattern on the mask plate is transferred to the second insulating layer, a plurality of pores corresponding to the electrode are formed in the second insulating layer, and an oil storage region is formed at the same time; finally, the bottom of the pore is subjected to a hydrophilic modification treatment to form a hydrophilic modification region at the bottom of the pore, thereby obtaining a nanopore sequencing chip.

19. The method of claim 18, wherein the nanopore sequencing chip is prepared by, The photoresist surface and the electrode are located on the same surface of the first insulating layer, and the light transmission area of the mask plate for forming the oil storage region is less than or equal to the structure size of the second insulating layer. The second insulating layer is prepared by spin-coating a layer of photoresist and exposing the photoresist once, and the upper layer and the lower layer of the second insulating layer present different structures.

20. The method for preparing the nanopore sequencing chip as described in claim 18, characterized in that, After the hydrophilic modification treatment, the upper end of the pore and the surface of the second insulating layer are further subjected to a hydrophobic modification treatment, so that a hydrophobic modification region is formed at the upper end of the pore and the surface of the second insulating layer.

21. A packaging structure of a nanopore sequencing chip, characterized by, The method comprises the nanopore sequencing chip according to claim 10.

22. The packaging structure of a nanopore sequencing chip according to claim 21, wherein, Also include a first plastic package, a packaging frame and a first metal lead, the first plastic package is provided with a first through hole, the packaging frame is installed at the bottom of the first through hole, and the nano-pore sequencing chip card is arranged in the packaging frame; the upper end of the first through hole and the upper surface of the nano-pore sequencing chip form a first liquid storage tank, and the first liquid storage tank is communicated with the arrayed holes.

23. The packaging structure of a nanopore sequencing chip according to claim 22, wherein, The chip pad is arranged on the side surface of the nano-pore sequencing chip, and the electrode wire is arranged in the nano-pore sequencing chip; the packaging frame is provided with a plurality of packaging frame pads corresponding to the chip pad on the side surface; The first metal lead is provided with a plurality of first metal leads corresponding to the chip pad, one end of each first metal lead is connected with the chip pad, and the other end is connected with the packaging frame pad.

24. The packaging structure of a nanopore sequencing chip according to claim 22, wherein, The first through hole is provided as a window, and the window is in the shape of a quadrilateral, a circle or an irregular figure in a plan view; the upper surface pattern and the lower surface pattern of the window are consistent or inconsistent; the side wall is perpendicular or non-perpendicular, or the perpendicular and non-perpendicular are alternately combined.

25. The packaging structure of a nanopore sequencing chip according to claim 22, wherein, The first through hole is provided as a window, and the window is provided as a cuboid, an inverted cone or an irregular groove.

26. The packaging structure of a nanopore sequencing chip according to claim 22, wherein, The bottom outer side of the plastic package is surrounded by a plurality of first metal contacts corresponding to the packaging frame pad, and the first metal contact and the packaging frame pad are electrically connected.

27. The packaging structure of a nanopore sequencing chip according to claim 21, wherein, Also include a second plastic package, a packaging substrate, a polymer gasket and a second metal lead; the nano-pore sequencing chip is arranged on the packaging substrate, the packaging substrate is arranged in the second plastic package, and the polymer gasket is arranged between the nano-pore sequencing chip and the second plastic package.

28. The packaging structure of a nanopore sequencing chip according to claim 27, wherein, The chip pad is arranged on the side surface of the nano-pore sequencing chip, and the electrode wire is arranged in the nano-pore sequencing chip; the packaging substrate is provided with a substrate pad corresponding to the chip pad on one side of the nano-pore sequencing chip, and is provided with an arrayed second metal contact on the other side; the second metal lead is arranged in the packaging substrate, one end of the second metal lead is connected with the substrate pad, and the other end is connected with the second metal contact; one end of the second metal lead connected with the substrate pad extends to the outside of the packaging substrate and is connected with the chip pad; The polymer gasket is provided with a quadrilateral or elliptical second through hole; the second through hole is arranged corresponding to the arrayed holes on the nano-pore sequencing chip.

29. The packaging structure of a nanopore sequencing chip according to claim 28, wherein, The second plastic package includes an upper shell and a bottom shell, the nano-pore sequencing chip is located between the upper shell and the bottom shell, and the front surface of the nano-pore sequencing chip faces the upper shell; The upper shell is provided with a liquid inlet, a second liquid storage tank, a waste liquid tank, a flow channel and a valve, the second liquid storage tank and the waste liquid tank are located at the bottom of the upper shell, the flow channel is used for connecting the second liquid storage tank and the waste liquid tank, and the valve is located at the top of the upper shell; the valve is used for controlling whether the flow channel is connected or not; The bottom shell is provided with a substrate card slot, and the packaging substrate is arranged in the substrate card slot; The upper shell and the bottom shell are detachably connected.

30. The packaging structure of a nanopore sequencing chip according to claim 29, wherein, The packaging base is further provided with an integrated circuit module and a heat conduction module penetrating the packaging base, the integrated circuit module is arranged on the side of the heat conduction module, and the nanopore sequencing chip is arranged on the heat conduction module; a data transmission interface is arranged on the side of the packaging base, a third through hole corresponding to the data transmission interface is arranged on the bottom shell, the data transmission interface is arranged in the third through hole and extends to the outside of the second plastic sealing shell.

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