Nanopore sequencing chip and preparation method and packaging structure thereof

Abstract

The invention discloses a nanopore sequencing chip as well as a preparation method and a packaging structure thereof, and relates to the fields of micro-nano processing, advanced packaging and biomedical treatment. The substrate comprises a first insulating layer, a second insulating layer and an electrode, the second insulating layer is arranged on the first insulating layer, and a plurality of holes arranged in an array are formed in the second insulating layer; the hole penetrates through the second insulating layer and is communicated with the first insulating layer; the electrode is arranged at the bottom of the hole; the second insulating layer is provided with a hydrophobic area, and the hydrophobic area is located above the hole and surrounds the hole. Hydrophilic modification areas are arranged at the bottoms of the holes. The preparation method is simple, and a similar double-layer structure can be prepared only through a one-time photoetching exposure technology by utilizing the light diffraction characteristic. The packaging structure adopts a metal lead and a pin-free packaging mode, so that the crosstalk problem of the metal lead is solved; separated transportation is adopted, and the shelf life can be prolonged. The nanopore sequencing chip is simple in structure, low in cost and good in stability.

Description

Technical Field

[0001] The present invention relates to the fields of micro-nano processing, advanced packaging and biomedicine, and in particular to a nanopore sequencing chip and a preparation method and packaging structure thereof. Background Art

[0002] Nanopore sequencing technology has the characteristics of long read length, low cost, high throughput, and high integration. It has a wide range of applications in biological fields such as molecular detection and gene sequencing.

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

[0004] The nanopore sequencing chip includes a photoresist layer and a hole array arranged in the photoresist layer. Each individual hole in the hole array is an independent sequencing unit. Therefore, the number and stability of the thin film formed in the hole array on a single chip are crucial to the flux and quality of the detection. At the same time, the cost of the nanopore sequencing chip is directly related to the terminal price of the nanopore sequencing application, which in turn 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 in patent CN115803624A. These structures are complex in shape, and the processing cost is high and difficult. In addition, due to the small pore size and the hydrophobicity of the photoresist layer, these characteristics cause the bottom of the pore to be hydrophobic, and it is difficult for the ionic solution to infiltrate the bottom of the pore and conduct with the electrode, resulting in poor pore wettability and low conductivity, thereby affecting the film formation rate and the stability of the film after film formation. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a nanopore sequencing chip and a preparation method and packaging structure thereof. The nanopore sequencing chip of the present invention has low cost, high yield, good structural wettability, high conductivity, simple preparation method, fast film formation and good stability of the film after film formation.

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

[0007] In a first aspect, the present invention 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 array-arranged holes are provided on the second insulating layer; the holes penetrate the second insulating layer and are connected to the first insulating layer; the electrodes are arranged at the bottom of the holes; a hydrophobic region is provided on the second insulating layer, the hydrophobic region is located above the holes and is arranged around each hole, the hydrophobic region is conducive to the formation and fixation of the membrane and the storage of organic solvents; the second insulating layer and the hydrophobic region form a double-layer insulating layer pore sequence structure; a hydrophilic modified region is provided at the bottom of the holes; the hydrophilic modified region is obtained by modification with a hydrophilic modified solution, and the hydrophilic modified solution is selected from one of a chitosan solution, a dopamine solution and a polylysine solution.

[0008] Furthermore, the holes arranged in the array are separated from each other by a second insulating layer medium, and the shape of the holes can be circular or irregular.

[0009] Furthermore, the shape of the electrode may be circular or polygonal, and the geometric center of the shape of the electrode coincides with the geometric center of the shape of the hole on the second insulating layer.

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

[0011] Furthermore, the nanopore sequencing chip also includes a signal transmission component, and the signal transmission component is connected to the electrode for transmitting a test signal.

[0012] Furthermore, the signal transmission component includes 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 to the electrode through the electrode line.

[0013] Preferably, the hydrophobic area includes an oil storage area. The oil storage area can form a quasi-double-layer insulation layer structure with the second insulation layer. By designing a honeycomb-shaped quasi-double-layer insulation layer structure, the stress generated by the photoresist during micro-nano processing can be reduced, the setting of the peripheral oil storage area on the chip can be eliminated, 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 area and the edge of the hole is 3 μm-50 μm.

[0015] Preferably, the height of the oil storage area is 1 μm-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 made of a photosensitive insulating material. Preferably, the photosensitive material is selected from one of photoresist, UV glue and photoresist film.

[0018] Preferably, the surface of the second insulating layer is provided with a plurality of boss structures or a plurality of groove structures, and the spaces between the plurality of boss structures or the plurality of groove structures form an oil storage area. The second insulating layer and the plurality of boss structures or the plurality of groove structures on its surface form a quasi-double-layer insulating layer pore structure that can improve film formation stability.

[0019] Furthermore, the shape of the boss structure may be circular, quasi-circular or polygonal, and the polygon includes a regular polygon and an irregular polygon.

[0020] Preferably, the shape of the boss structure is hexagonal, and a plurality of the hexagonal boss structures are regularly arranged on the second insulating layer. Preferably, the margin of the hexagonal boss structure is 1 μm-50 μm.

[0021] Preferably, a plurality of the hexagonal boss structures form regular channels, and the regular channels and the hexagonal boss structures form a honeycomb structure. Preferably, the depth of the regular channels is 1 μm-50 μm.

[0022] Furthermore, the shape of the groove structure is circular or irregular polygonal, and the plurality of groove structures are regularly arranged to form an oil storage area. Preferably, the depth of the groove structure is 1 μm-50 μm.

[0023] Preferably, the hole is a circular hole structure, the diameter of the hole is 20 μm-150 μm, and the height of the hole is 10 μm-150 μm.

[0024] Preferably, the hydrophobic region comprises a hydrophobic modified region, and the hydrophobic modified region is obtained by hydrophobic modification with a hydrophobic modified solution, and the hydrophobic modified solution is selected from one of fluorine-containing compounds, polysilicon compounds, and synthetic carbon chain compounds.

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

[0026] Furthermore, the hydrophobic modification comprises the following steps:

[0027] The hydrophobic modified solution is added dropwise at the geometric center of the surface of the second insulating layer, so that the hydrophobic modified solution spreads to the surroundings, covers the upper surface of the second insulating layer and the upper end of the hole without penetrating into the bottom of the hole; and then heated at a temperature of 100°C-200°C for 30min-180min to form a hydrophobic area above the hole.

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

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

[0030] Fluorine-containing compounds are added dropwise to the heating device, and evaporated into gaseous form under high temperature. High-purity nitrogen or inert gas is used to drive the gaseous fluorine-containing compounds to fill the modified environment and evenly deposit on the surface of the second insulating layer to form a hydrophobic area.

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

[0032] During the modification process, the sample carrying device drives the sample to be modified to rotate at a constant speed, the air inlet continuously supplies heated nitrogen or inert gas, and the heating device can quickly control the temperature change.

[0033] Furthermore, since the pore size is small and the photoresist is a hydrophobic material, it is difficult for polar liquids to infiltrate the bottom of the pores, and vacuum or other gas replacement methods are usually required to infiltrate and conduct them. For example, Chengdu Qi Tan Technology Co., Ltd. disclosed a technical solution for conducting the pores using carbon dioxide replacement technology in patent CN215757392U; in addition, hydrophilic modification of the bottom of the pores can make the nanopore sequencing chip able to be infiltrated and conducted in the absence of vacuum or other gas replacement methods, but it is difficult to partially hydrophilize the pores.

[0034] Therefore, the present invention provides a method for preparing the hydrophilic modified region in the above-mentioned nanopore sequencing chip, comprising the following steps:

[0035] S1. Hydrophilic modification: Infiltrate the wells with a hydrophilic modification solution having a mass concentration of 5%-20%, and modify for 30min-180min at a temperature of 20°C-50°C; if the concentration of the hydrophilic modification solution is greater than the range of this application, floccules are likely to precipitate on the chip surface during solution modification, increasing the difficulty of subsequent cleaning, and affecting the infiltration effect in the wells, thereby affecting the modification effect;

[0036] S2, removing the hydrophilic modification solution at the hole mouth and retaining the hydrophilic modification solution at the bottom of the hole, so that the upper end of the hole returns to a hydrophobic state; then aging it for more than 5 days, so that a hydrophilic modification area is formed at the bottom of the hole under the action of the hydrophilic modification solution.

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

[0038] Furthermore, the specific steps of removing the hydrophilic modification solution at the orifice and retaining the hydrophilic modification solution at the bottom of the orifice are as follows:

[0039] The surface of the hole sequence structure is cleaned with pure water and wiped dry, and then it is rotated at a constant speed under vacuum adsorption conditions and its surface is rinsed 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 cleaned and the hydrophilic modification solution remains at the bottom of the hole; the water on the surface is then blown dry with an air gun to remove the moisture on the surface of the second insulating layer and keep the hole moist.

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

[0041] Furthermore, in the step S2, after aging, the chip is cleaned again with a high-pressure water gun to remove crystals, and then blow-dried and heated to dry the moisture.

[0042] In a second aspect, the present invention also provides another nanopore sequencing chip, which is based on the nanopore sequencing chip described in the first aspect, and the signal transmission component of the nanopore sequencing chip further includes an integrated circuit area and a first electrode line, wherein the integrated circuit area is disposed on the first insulating layer and is connected to the electrode through the first electrode line. One end of the electrode line is connected to the chip pad, and the other end is connected to the electrode through the integrated circuit area and the first electrode line.

[0043] Furthermore, the first insulating layer includes a base layer and an insulating layer, and the insulating layer is arranged between the second insulating layer and the base layer; and the integrated circuit area is located between the base layer and the insulating layer.

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

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

[0046] Furthermore, the base layer includes a single crystal silicon layer, and the integrated circuit region is arranged between the single crystal silicon layer and the insulating layer.

[0047] Preferably, the base 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 includes 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 connected in sequence through a fourth electrode line.

[0049] Preferably, after the electrode receives the signal, the current signal can be transmitted 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 current signals of multiple electrodes are received simultaneously through a multiplexer, and then the current signal is transmitted to the analog-to-digital converter through the fourth electrode line. The analog-to-digital converter can convert the current signal into a digital signal and output it.

[0050] Preferably, there are a plurality of integrated circuit areas.

[0051] In a third aspect, the present invention further provides a method for preparing a nanopore sequencing chip as described in the first aspect, which utilizes the diffraction characteristics of light and can process a nanopore sequencing chip into a quasi-double-layer insulating layer pore structure through a single photolithography exposure technology. The quasi-double-layer insulating layer honeycomb structure in the present invention can not only form a hydrophobic structure similar to the Wenzel-Cassie model, making the surface present a strong hydrophobicity, but also the honeycomb structure can act as a stress release area, thereby eliminating the need to set a stress release functional area on the chip, saving chip area and reducing costs; the method comprises the following steps:

[0052] Electrodes are arranged on the first insulating layer, and then 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 is transferred to the second insulating layer, and a plurality of holes corresponding to the electrodes are formed on the second insulating layer, and an oil storage area is formed at the same time; finally, the bottom of the hole is hydrophilically modified to form a hydrophilic modified area at the bottom to obtain a nanopore sequencing chip.

[0053] Furthermore, the photoresist surface and the electrode are both located on the same surface of the first insulating layer, and the light-transmitting area of ​​the oil storage area made by the mask is smaller than or equal to the structural 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, that is, a quasi-double-layer structure.

[0055] Preferably, the surface of the nanopore sequencing chip can be hydrophobically modified after the hydrophilic modification, thereby forming a hydrophobic modified area at the upper end of the hole and the surface of the second insulating layer. The hydrophobic modified area can further enhance the hydrophobic properties of the second insulating layer and the top of the hole, improve its film forming rate and the stability of the film after film formation. The hydrophobic modification is achieved by regulating the composition and dosage of the hydrophobic modification solution so that the hydrophobic modification solution does not enter / or enters less into the bottom of the hole, or the hydrophobic modification solution does not physically / chemically react with the hydrophilic layer, thereby forming an asymmetric hydrophilic-hydrophobic structure in which the bottom of the hole is hydrophilic and the top of the hole and the surface of the second insulating layer are hydrophobic.

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

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

[0058] Furthermore, 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] Furthermore, when the exposure mode is hard contact or vacuum contact, the exposure dose is 1.5 to 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. Traditional nanopore sequencing chips are bonded and packaged with PC plastic or acrylic shells, which is costly and has poor sealing; 2. Nanopore sequencing chips are large in size and are bonded to circuit boards through ball grid array packaging (BGA), which has high equipment requirements, cumbersome steps, high costs, and low yields; 3. Sequencing chips are transported in a way that protein pores, buffer solutions, and chips are concentrated together, with a limited shelf life, which makes the shelf life of the chip extremely short; 4. Some sequencing chips on the market require stress release areas to be designed on the bare chip, and the packaging area is too large, increasing costs.

[0061] Therefore, in a fourth aspect, in order to solve the above-mentioned problem, the present invention provides a packaging structure suitable for the nanopore sequencing chip described in the first aspect, comprising the nanopore sequencing chip described in the first aspect, a first plastic package shell, a packaging frame and a first metal lead.

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

[0063] The first through hole may be a window, and the shape of the window in top view may be a quadrilateral, a circle or an irregular shape; the upper surface shape and the lower surface shape of the window may be consistent / or inconsistent; the side wall may be set to be vertical / or non-vertical, or an alternating combination of vertical and non-vertical.

[0064] The window can be configured as a cuboid, a through hole, an inverted frustum, or an irregular groove.

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

[0066] The signal transmission component is a first signal transmission component, and the first signal transmission component 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 of the nanopore sequencing chip, the second electrode line is arranged inside the nanopore sequencing chip, and the first chip pad is connected to the electrode through the second electrode line; a plurality of packaging frame pads corresponding to the first chip pad are arranged around the side of the packaging frame; the first metal lead is located above the nanopore sequencing chip; each of the first chip pads and the packaging frame pad is connected through the first metal lead.

[0067] A plurality of first metal contacts are arranged around the outer side of the bottom of the plastic-sealed shell, and the first metal contacts are used for connecting and conducting electricity.

[0068] Furthermore, a pore sequencing region is formed between the pores of the nanopore sequencing chip, and the pore sequencing region is completely exposed in the liquid storage tank to further ensure the film formation rate of the nanopore sequencing chip.

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

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

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

[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] Furthermore, the number of the first metal contacts is selected from 1 to 120; the number of the first metal contacts, the first chip pads and the package frame pads all correspond to the number of the holes. Preferably, the package frame is a rectangular or circular structure; the shape of the bottom of the first plastic package shell is consistent with the shape of the package frame.

[0074] In a fifth aspect, the present invention further provides a packaging structure applicable to the nanopore sequencing chip of the second aspect that can solve the above problem, and the packaging structure is different from the packaging structure of the fourth aspect in that:

[0075] It comprises the nanopore sequencing chip as described in the second aspect, a first plastic package shell, a packaging frame and a first metal lead.

[0076] The signal transmission component is a second signal transmission component, and the second signal transmission component includes 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, and the third electrode line is arranged inside the nanopore sequencing chip. The second chip pad is connected to the integrated circuit area through the third electrode line. The second chip pad is connected to the package frame pad through the first metal lead. The number of the first metal contact, the second chip pad, and the package frame pad is set accordingly.

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

[0078] Furthermore, the number of holes in the nanopore sequencing chip in the package structure does not need to correspond one-to-one with the number of pads on the second chip, and it can be n times the number of pads on the second chip, where n is a positive integer. Therefore, the number of pads required for the second chip can be much less than the number of holes, thereby increasing the density and number of holes on a single chip, as well as the sequencing throughput, further reducing the volume and reducing the sequencing cost. In addition, the package structure can directly convert the real-time measured data through the integrated circuit area and output it through the second chip pad, which can reduce the interference generated during signal transmission.

[0079] In a sixth aspect, the present invention also provides another packaging structure that can solve the above-mentioned problem and is suitable for the nanopore sequencing chip described in the first aspect, comprising the nanopore sequencing chip described in the first aspect, a second plastic packaging shell, a packaging substrate, a polymer gasket and a second metal lead.

[0080] The nanopore sequencing chip is arranged on a packaging substrate, the signal transmission component is a first signal transmission component, the first signal transmission component includes a first chip pad and a second electrode line, the first chip pad is arranged on the side of the nanopore sequencing chip, the second electrode line is arranged inside the nanopore sequencing chip, and the second electrode line connects the first chip pad and the electrode; the packaging substrate is arranged inside the second plastic package shell, and the polymer gasket is arranged between the nanopore sequencing chip and the second plastic package shell. The upper shell is provided with a liquid inlet, a second liquid reservoir, a waste liquid tank, a flow channel and a valve, the liquid reservoir and the waste liquid tank are located at the bottom of the upper shell, the flow channel is used to connect the second liquid reservoir and the waste liquid tank, and the valve is located at the top of the upper shell; the valve is used to control whether the flow channel is connected. The packaging substrate does not contain a high-density integrated circuit; the packaging substrate is provided with a substrate pad corresponding to the first chip pad on one side of the nanopore sequencing chip, and an array-arranged second metal contact is provided on the other side; the second metal lead is passed through the interior of the packaging substrate, one end is connected to the substrate pad, and the other end is connected to the second metal contact. The substrate pad is located outside the nanopore sequencing chip, and one end of the second metal lead connected to the substrate pad extends to the outside of the packaging substrate and is connected to the first chip pad.

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

[0082] Furthermore, the second plastic-sealed shell includes an upper shell and a bottom shell, the nanopore sequencing chip is located between the upper shell and the bottom shell, and the front side of the nanopore sequencing chip is arranged toward the upper shell.

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

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

[0085] Preferably, the packaging structure of the nanopore sequencing chip also includes a threaded structure, and a first threaded hole is provided on the side of the upper shell; a second threaded hole corresponding to the first threaded hole is provided on the side of the bottom shell, and the threaded structure is penetrated into the first threaded hole and the second threaded hole, and the upper shell and the bottom shell are fixedly connected by threads.

[0086] Furthermore, a quadrilateral or elliptical second through hole is provided inside the polymer gasket; the second through hole is arranged corresponding to the holes arranged in an array on the nanopore sequencing chip.

[0087] Furthermore, the bottom shell is placed in the lower layer, and the bottom shell is bonded to the side of the packaging substrate where the second metal contact is set; the side of the packaging substrate where the substrate pad is set is bonded 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 the substrate pads is selected from one of 120-512; the number of the substrate pads, the first chip pads and the second metal contacts corresponds to the number of the holes.

[0089] In a seventh aspect, the present invention also provides another packaging structure that can solve the above-mentioned problem and is applicable to the nanopore sequencing chip described in the first aspect, which is different from the packaging structure described in the sixth aspect as follows:

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

[0091] In an eighth aspect, the present invention also provides another packaging structure suitable for the nanopore sequencing chip described in the second aspect, which includes the nanopore sequencing chip described in the second aspect, a second plastic packaging shell, a packaging substrate, a polymer gasket and a second metal lead.

[0092] The difference between the sixth aspect and the sixth aspect is that:

[0093] The signal transmission component is a second signal transmission component, and the second signal transmission component includes 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 a second metal lead. The number of the second metal contact, the second chip pad, and the package frame pad is set accordingly.

[0094] Furthermore, the packaging structure can directly convert the real-time measured data through the integrated circuit area and output it 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 increasing the density and number of holes on a single chip, as well as the sequencing throughput, further reducing the volume and reducing the sequencing cost.

[0095] In summary, the metal leads and square flat pinless packaging in the present invention are suitable for nanopore sequencing chips with a variety of hole numbers, which can reduce the volume and cost, and at the same time have good sealing performance and reduce the crosstalk problem caused by metal leads; and the nanopore sequencing chip can be transported separately. During the transportation process, the nanopore sequencing chip will not come into contact with any liquid, which can effectively improve the shelf life of the nanopore sequencing chip.

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

[0097] Furthermore, the nanopore sequencing chip of the present invention, combined with the above-mentioned packaging method and membrane laying method, can be laid out and used immediately and produce experimental data within 30 minutes.

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

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

[0100] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0101] Figure 1 Schematic diagram of a single pore structure of the nanopore sequencing chip provided in Example 1 of the present invention.

[0102] Figure 2 This is a front schematic diagram of a single pore structure of the nanopore sequencing chip provided in Example 1 of the present invention.

[0103] Figure 3 for Figure 2 AA cross-section diagram.

[0104] Figure 4 This is a schematic diagram of the physical structure of a single pore of the nanopore sequencing chip provided in Example 1 of the present invention.

[0105] Figure 5 This is a schematic diagram of the hydrophilic modification provided in Example 1 of the present invention.

[0106] Figure 6Schematic diagram of the hydrophilic modification solution for removing the pore surface provided in Example 1 of the present invention.

[0107] Figure 7 Schematic diagram of the packaging structure of the nanopore sequencing chip provided in Example 1 of the present invention.

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

[0109] Fig. 9 This is a schematic diagram of the front structure of the packaging structure of the nanopore sequencing chip provided in Example 1 of the present invention in the shell-less state.

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

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

[0112] Fig.12 Schematic diagram of ion solution conduction of the nanopore sequencing chip provided in Example 1 of the present invention.

[0113] Fig.13 Schematic diagram of membrane laying for the nanopore sequencing chip provided in Example 1 of the present invention.

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

[0115] Fig.15 This is a front schematic diagram of a single pore structure of the nanopore sequencing chip provided in Example 2 of the present invention.

[0116] Fig.16 for Fig.15 AA cross-section diagram.

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

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

[0119] Fig.19 Schematic diagram of a single hole structure of the hole sequence provided in Example 3 of the present invention.

[0120] Fig. 20 A cross-sectional view of a single pore structure of the pore sequence provided in Example 3 of the present invention.

[0121] Fig.21Schematic diagram of the hydrophobic modification of the nanopore sequencing chip provided in Example 3 of the present invention.

[0122] Fig. 22 This is an exploded view of the packaging structure of the nanopore sequencing chip provided in Example 3 of the present invention.

[0123] Fig.23 Schematic diagram of the packaging structure of the nanopore sequencing chip provided in Example 3 of the present invention.

[0124] Fig.24 This is a schematic diagram of the bottom structure of the upper shell provided in Example 3 of the present invention.

[0125] Fig.25 This is a schematic diagram of the front structure of the packaging substrate provided in Example 3 of the present invention.

[0126] Fig.26 This is a schematic diagram of the back structure of the packaging substrate provided in Example 3 of the present invention.

[0127] Fig. 27 A physical picture of the packaging structure of the nanopore sequencing chip provided in Example 3 of the present invention.

[0128] Fig.28 This is a schematic diagram of hydrophobic modification provided in Example 4 of the present invention.

[0129] Fig.29 This is a schematic diagram of the packaging substrate structure provided in Example 4 of the present invention.

[0130] Fig.30 This is a diagram showing the overall structure of the nanopore sequencing chip provided in Example 5 of the present invention.

[0131] Fig.31 A top view of the nanopore sequencing chip provided in Example 5 of the present invention.

[0132] Fig.32 This is a cross-sectional view of the nanopore sequencing chip provided in Example 5 of the present invention.

[0133] Fig.33 Another cross-sectional view of the nanopore sequencing chip provided in Example 5 of the present invention.

[0134] Fig.34 This is a diagram showing the overall structure of the nanopore sequencing chip provided in Example 7 of the present invention.

[0135] Fig.35 This is a cross-sectional view of the nanopore sequencing chip provided in Example 7 of the present invention.

[0136] Fig.36 This is a schematic diagram of the signal transmission principle of the nanopore sequencing chip provided in Example 5 of the present invention.

[0137] Fig.37 Schematic diagram of the packaging structure of the nanopore sequencing chip provided in Example 8 of the present invention.

[0138] Fig.38 This is a diagram of the water droplet state on the hole surface provided for comparative example 1 of the present invention.

[0139] Fig.39 This is a diagram of the water droplet state on the hole surface provided for Comparative Example 2 of the present invention.

[0140] Fig.40 This is a diagram of the water droplet state on the hole surface provided in Example 3 of the present invention.

[0141] Fig.41 Schematic diagram of the conduction effect of the nanopore sequencing chip provided in Example 1 of the present invention.

[0142] Fig.42 Schematic diagram of the film-forming effect of the nanopore sequencing chip provided in Example 1 of the present invention.

[0143] Fig.43 Schematic diagram of the sequencing effect of the nanopore sequencing chip provided in Example 1 of the present invention.

[0144] Description of the symbols in the figure:

[0145] 1-first insulating layer; 101-single crystal silicon layer; 102-glass layer; 103-insulating layer; 2-second insulating layer; 201-hole; 202-hydrophilic modified area; 3-hydrophobic modified solution; 4-electrode; 5-hydrophilic modified solution; 6-boss structure; 7-groove structure; 8-packaging frame; 9-nanopore sequencing chip; 901-hole sequencing area; 902-first chip pad; 903-second chip pad; 904-third electrode line; 10-plastic sealing structure; 11-packaging frame pad; 12-first metal lead; 13-first metal contact; 14-first plastic sealing shell; 15-first liquid storage tank; 16-pit structure; 17-rectangular teeth; 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-substrate slot;222-second threaded hole;23-package substrate;24-polymer gasket;25-second metal contact;26-substrate pad;27-second through hole;28-air inlet;29-exhaust port;30-glue tray;31-fluorine-containing compound;32-heating table;33-integrated circuit module;34-thermal conductive module;35-integrated circuit area;3501-signal amplifier;3502-multiplexer;3503-analog-to-digital converter;36-first electrode line;37-data transmission interface. DETAILED DESCRIPTION

[0146] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0148] In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings, or are the directions or positional relationships in which the inventive product is usually placed when in use. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as a limitation on the present invention.

[0149] In the description of the present invention, it is also necessary to explain that, unless otherwise clearly specified and limited, the terms "set", "install", "connect", and "connect" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection, or an indirect connection through an intermediate medium, or it can be the internal communication of two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0150] In an embodiment of the present invention, the specific steps of removing the hydrophilic modification solution on the surface of the quasi-double-layer insulating layer pore structure and retaining the hydrophilic modification solution at the bottom of the pore are as follows:

[0151] The surface of the quasi-double-layer insulation layer structure is cleaned with pure water and wiped dry, and then placed on the surface of the tray of the glue spreader, and the vacuum mode is turned on to make the back of the quasi-double-layer insulation layer hole sequence structure close to the surface of the tray. The glue spreader drives the quasi-double-layer insulation layer hole sequence structure to rotate slowly, and then rinse its surface 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 use an air gun to blow dry the water on its surface, so that the moisture on the surface of the second insulation layer is removed and the hole is kept moist; the angle between the muzzle of the high-pressure water gun and its surface is 15-60°; the angle between the muzzle of the air gun and its surface is maintained at 15-60°.

[0152] Embodiment 1:

[0153] Please refer to Figure 1-4 The nanopore sequencing chip shown includes a first insulating layer 1, a second insulating layer 2 and an electrode 4, wherein the second insulating layer 2 is arranged on the first insulating layer 1, and an array of holes 201 are provided on the second insulating layer 2; the holes 201 penetrate the second insulating layer 2 and are connected to the first insulating layer 1; the electrode 4 is arranged at the bottom of the hole 201; a hydrophilic modified area 202 is provided at the bottom of the hole 201; the hydrophilic modified area 202 is obtained by modification with a hydrophilic modified solution, and the hydrophilic modified solution is a chitosan solution; a hydrophobic area is provided on the second insulating layer 2, and in this embodiment, the hydrophobic area is an oil storage area, and the oil storage area is arranged around each hole 201.

[0154] Specifically, a plurality of hexagonal boss structures 6 are provided on the upper surface of the second insulating layer 2, and the spaces between the plurality of hexagonal boss structures 6 form regular flow channels, and the regular flow channels and the hexagonal boss structures 6 form a honeycomb-like oil storage area. The regular flow channels can guide the ion solution 19 to flow back into the pores 201, so that a stable oil-in-water structure is formed in the pores 201. The second insulating layer 2 and the honeycomb-like oil storage area can form a pore sequence structure similar to a double-layer insulating layer.

[0155] In this embodiment, multiple hexagonal boss structures 6 can support the oily film on the hole surface, and the honeycomb-like oil storage area can improve the hydrophobicity of the surface of the second insulating layer 2, which is beneficial to the dredging 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 larger than the aperture of the hole 201 to ensure the contact area between the electrode 4 and the ion solution 19 .

[0158] For more details, please refer to Fig. 9 The nanopore sequencing chip 9 also includes a first signal transmission component, which includes 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, and the second electrode line is arranged inside the nanopore sequencing chip 9. The first chip pad 902 is connected to the electrode 4 through the second electrode line.

[0159] Specifically, the method for preparing the nanopore sequencing chip of this embodiment includes the following steps:

[0160] S1, an electrode 4 is arranged on the first insulating layer 1, and then a photoresist is coated on the upper surface of the first insulating layer 1. According to the diffraction characteristics of light, a second insulating layer 2 is prepared on the first insulating layer 1 by photolithography exposure, and a hole 201 corresponding to and connected to the electrode 4 is formed on the second insulating layer 2, and a hexagonal prism boss arranged around the top of the hole 201 is formed, and then a honeycomb-like oil storage area is formed on the second insulating layer 2, so that a quasi-double-layer insulating layer hole sequence structure can be formed by a single photolithography exposure;

[0161] S2, hydrophilic modification; e.g. Figure 5 As shown, the quasi-double-layer insulating layer hole sequence structure is modified by immersing in a hydrophilic modification solution 5 with a mass concentration of 5%, so that the hydrophilic modification solution 5 infiltrates the hole 201, and the modification is carried out at a temperature of 30° C. for 60 minutes to obtain a hydrophilic modification solution layer on the surface of the quasi-double-layer insulating layer hole sequence structure.

[0162] S3, such as Figure 6 As shown, part of the hydrophilic modification solution 5 on the surface of the quasi-double-layer insulating layer pore sequence structure is removed and the hydrophilic modification solution 5 at the bottom of the pore 201 is retained, so that the upper surface of the quasi-double-layer insulating layer structure and the mouth of the pore 201 are restored to a hydrophobic state; after aging for 6 days, a hydrophilic modification area 202 is formed at the bottom of the pore 201 under the action of the hydrophilic modification solution 5, and finally, it is cleaned with a high-pressure water gun to remove crystals, blown dry and heated to dry the moisture, and a nanopore sequencing chip is obtained.

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

[0164] The surface of the quasi-double-layer insulation layer structure is cleaned with pure water and wiped dry, and it is placed on the surface of the glue spreader tray. The vacuum mode is turned on to allow the opposite side of the quasi-double-layer insulation layer structure to be tightly attached to the surface of the tray. The glue spreader drives the quasi-double-layer insulation layer structure to rotate slowly at a speed of 100r / s, and then rinses its surface with a high-pressure water gun until only the hydrophilic modified solution 5 remains at the bottom of the hole 201; then, the water on its surface is blown dry with an air gun to remove the moisture on the surface of the second insulation layer 2 and keep the hole 201 moist; the angle between the muzzle of the high-pressure water gun and its surface is 15°; the angle between the muzzle of the air gun and its surface is maintained at 60°.

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

[0166] Specifically, the honeycomb-like structure on the surface of the nanopore sequencing chip prepared by this embodiment is relatively small in size, which can effectively improve the hydrophobicity of the surface of the second insulating layer 2, and can further improve the stability of the film after film formation through the oil storage area. Secondly, the nanopore sequencing chip of this embodiment can obtain a quasi-double-layer insulating layer pore sequence structure through a single photolithography exposure, which is simple and reliable, with a short processing time and low cost. It does not require a high-resolution mask and a high-end lithography machine, and can be manufactured and processed using an ordinary exposure method of an ordinary lithography machine and ordinary photoresist.

[0167] Please refer to further Figure 7-11 This embodiment also provides a packaging structure of a nanopore sequencing chip, including a plastic packaging structure 10 and the above-mentioned nanopore sequencing chip 9, wherein the plastic packaging structure 10 includes 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 installed at the bottom of the first through hole. The packaging frame 8 is used to carry the nanopore sequencing chip 9 and dissipate heat to achieve a temperature control effect; the nanopore sequencing chip 9 is clamped inside the packaging frame 8; and a first liquid storage tank 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 the package frame pad 11 are connected by the first metal lead 12; and a first metal contact 13 for connecting to conduction is arranged around the outside of the bottom of the first plastic package shell 14. 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 tank 15, so as to further ensure the film formation rate of the nanopore sequencing chip 9.

[0169] Specifically, in this embodiment, the number of holes 201 , first chip pads 902 , package frame pads 11 and first metal contacts 13 are all 96, and the package frame 8 is a metal frame.

[0170] Specifically, in this embodiment, metal leads and square flat pinless packaging are used for packaging, which can reduce costs and have good sealing, reducing crosstalk problems caused by metal leads; and the nanopore sequencing chip does not integrate functional modules such as signal processing circuits; the nanopore sequencing chip is easy to clean and has a high reuse rate, reducing the user's sequencing cost.

[0171] Specifically, the membrane laying method of the nanopore sequencing chip in this embodiment includes the following steps:

[0172] (1) using a pipette to drip the ion solution 19 onto the surface of the nanopore sequencing chip 9, so that the ion solution 19 is completely filled in the liquid reservoir 15 of the plastic package structure 10. At this time, the ion solution 19 cannot infiltrate into the pore 201;

[0173] (2) The surface of the nanopore sequencing chip 9 is repeatedly pumped and discharged until the ionic solution 19 enters the pore 201, making the pore 201 conductive. Fig.12 As shown;

[0174] (3) Use a pipette to extract the organic solution and bubble it on the surface of the hole 201. Fig.13 As shown, the organic solution is fixed into an oily film 18 under the hydrophobic and lipophilic effects of the surface structure of the nanopore sequencing chip structure, so that the ionic solution 19 in the pore 201 is isolated from the ionic solution 19 outside the pore 201 .

[0175] Specifically, the nanopore sequencing chip of this embodiment has a simple membrane laying method, and can quickly perform operations such as conduction, membrane laying, and protein pore insertion, and data can be obtained within 30 minutes; the instant membrane laying method is adopted, and the service life is long;

[0176] Example 2

[0177] The difference between this embodiment and embodiment 1 lies in 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] See also Figure 14-18 In the nanopore sequencing chip shown, the oil storage area in this embodiment 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 quasi-double-layer insulating layer hole sequence structure; a pit structure 16 is arranged along the inner side of the top of the hole 201; the distance between the groove structure 7 and the edge of the hole 201 is 10μm; the height of the hole 201 is 50μm; the height of the oil storage area is 5μm. The groove structure 7 can improve the hydrophobicity of the surface of the second insulating layer 2 and support the membrane on the surface of the hole. At the same time, the groove structure 7 is conducive to the storage of organic solvents, thereby facilitating the formation and fixation of the oily film 18. Specifically, in this embodiment, the hydrophilic modified solution is a dopamine solution.

[0179] Specifically, in this embodiment, the method for preparing a nanopore sequencing chip includes the following steps:

[0180] S1, an electrode 4 is arranged on the first insulating layer 1, and then a photoresist is coated on the first insulating layer 1. According to the diffraction characteristics of light, a second insulating layer 2 is prepared on the first insulating layer 1 by photolithography exposure, and a hole 201 corresponding to and connected to the electrode 4 is formed on the second insulating layer 2, and a groove structure 7 arranged around the hole 201 is formed, and then a groove-type oil storage area is formed on the surface of the second insulating layer 2; thus, a quasi-double-layer insulating layer hole sequence structure can be formed by a single photolithography exposure;

[0181] S2, hydrophilic modification; the double-layer insulating layer pore sequence structure is modified by immersing it in a dopamine solution with a mass concentration of 10%, so that the dopamine solution infiltrates the hole 201, and the modification is carried out at a temperature of 20°C for 180 minutes to obtain a dopamine solution layer on the surface of the double-layer insulating layer pore sequence structure.

[0182] S3. Remove part of the dopamine solution layer on the surface of the double-layer insulating layer and retain the dopamine solution layer at the bottom of the pore 201, so that the upper surface of the pore sequence structure and the nanopore mouth are restored to a hydrophobic state; age for 6 days to form a hydrophilic modified area 202 at the bottom of the pore 201 under the action of the dopamine solution, and obtain a nanopore sequencing chip.

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

[0184] The surface of the quasi-double-layer insulation layer structure is cleaned with pure water and wiped dry, and the opposite side of the structure surface is placed on the surface of the glue spreader tray, and the vacuum mode is turned on to make the opposite side of the quasi-double-layer insulation layer structure close to the surface of the tray. The glue spreader drives the hole sequence structure to rotate slowly at a speed of 100r / s, and then rinse its surface 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 insulation layer is cleaned and the dopamine solution layer remains at the bottom of the hole 201; then use an air gun to blow dry the water on its surface, so that the moisture on the surface of the second insulation layer is removed and the hole 201 is kept moist; the angle between the muzzle of the high-pressure water gun and its surface is 30°; the angle between the muzzle of the air gun and its surface is maintained at 30°.

[0185] Specifically, the groove structure size of the nanopore sequencing chip prepared by this embodiment is relatively small, which can effectively improve the hydrophobicity of the surface of the second insulating layer 2, and can further improve the stability of the film after film formation through the oil storage area. Secondly, the nanopore sequencing chip of this embodiment can obtain a quasi-double-layer insulating layer pore sequence structure through a single photolithography exposure, which is simple and reliable, with a short processing time and low cost. It does not require a high-resolution mask and a high-end photolithography machine, and can be manufactured and processed using an ordinary exposure method of an ordinary photolithography machine and ordinary photoresist.

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

[0187] Example 3

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

[0189] In this embodiment, the hydrophobic area is a hydrophobic modified area. The area around the top of the hole 201 is a smooth plane 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 area, which can achieve a surface hydrophobic effect, thereby enhancing the adhesion between the surface of the second insulating layer 2 and the oily film 18, facilitating the formation and fixation of the film, and forming a stable oil-in-water structure in the hole 201.

[0190] Specifically, in this embodiment, the hydrophilic modified solution 5 is a poly-lysine solution.

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

[0192] S1, an electrode 4 is arranged on the first insulating layer 1, and then a photoresist is coated on the upper surface of the first insulating layer 1, and a second insulating layer 2 is prepared on the first insulating layer 1 by a first photolithography exposure, and a hole 201 corresponding to and connected to the electrode 4 is formed on the second insulating layer 2 to obtain a single-layer hole sequence structure, such as Fig.19 , Fig. 20 As shown;

[0193] S2, hydrophilic modification; the single-layer pore sequence structure is modified by soaking in a poly-lysine solution having a mass concentration of 8%, so that the poly-lysine solution infiltrates the pores 201, and the modification is carried out at a temperature of 50°C for 30 minutes;

[0194] S3, removing part of the poly-lysine solution on the surface of the structure modified by S2 and retaining the poly-lysine solution at the bottom of the hole 201, so that the surface is restored to a hydrophobic state; aging for 6 days, so that the bottom of the hole 201 forms a hydrophilic modified area 202 under the action of the poly-lysine solution, and obtaining a hydrophilic modified chip;

[0195] S4, hydrophobic modification, the hydrophilic modified chip obtained in step S3 is hydrophobically modified; a hydrophobic modification solution 3 is dripped on the center of the surface of the second insulating layer 2 by a dispensing machine, such as Fig.21 As shown; in this embodiment, the hydrophobic modified solution 3 is a polysilicon compound, and the concentration of the polysilicon compound is 10%-60%, especially, the polysilicon compound content is 29% in this embodiment; the volume of the polysilicon compound added is 0.3μL-5μL, especially, the volume of the polysilicon compound added in this embodiment is 0.5μL; the volume of the polysilicon compound needs to be adjusted accordingly according to the changes in the diameter, height and number of the holes. If the polysilicon compound exceeds the range, it is easy for the polysilicon compound to infiltrate the entire hole 201, so that the hydrophilic modified area 202 at the bottom of the hole 201 and the electrode 4 are covered by the polysilicon compound, resulting in the hole 201 being unable 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 placed in a ventilated environment for heating and modified at a temperature of 150° C. for 30 minutes to form a hydrophobic modified area on the top of the hole 201 to obtain a nanopore sequencing chip.

[0197] Specifically, in this embodiment, the specific steps of removing part of the poly-lysine solution on the surface of the single-layer pore sequence structure and retaining the poly-lysine solution at the bottom of the pores are as follows:

[0198] The surface of the hole sequence structure is cleaned with pure water and wiped dry, and the opposite side of the structure surface is placed on the surface of the glue spreader tray. The vacuum mode is turned on to make the opposite side of the hole sequence structure surface of the double-layer insulation layer close to the tray surface. The glue spreader drives the hole sequence structure to rotate slowly at a speed of 100r / s, and then rinses its surface 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 use an air gun to blow dry the water on the surface, so that the moisture on the surface of the second insulation layer is removed and the hole is kept moist; the angle between the muzzle of the high-pressure water gun and its surface is 60°; the angle between the muzzle of the air gun and its surface is maintained at 15°.

[0199] For details, please refer to Figure 22-27 In this embodiment, the packaging structure of the nanopore sequencing chip includes the above-mentioned nanopore sequencing chip, a second plastic packaging shell, a packaging substrate 23 and a polymer gasket 24; the nanopore sequencing chip is arranged on the packaging substrate 23, the packaging substrate 23 is arranged inside the second plastic packaging shell, and the polymer gasket 24 is arranged between the nanopore sequencing chip and the second plastic packaging shell.

[0200] Specifically, the second plastic-sealed housing includes an upper housing 21 and a bottom housing 22, the nanopore sequencing chip is located between the upper housing 21 and the bottom housing 22, and the front of the nanopore sequencing chip is arranged toward the upper housing 21;

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

[0202] Specifically, it also includes a threaded structure. A first threaded hole 216 is provided on the side of the upper shell 21, and a second threaded hole 222 corresponding to the first threaded hole 216 is provided on the side of the bottom shell 22. The threaded structure is penetrated through the first threaded hole 216 and the second threaded hole 222, and the upper shell 21 and the bottom shell 22 are fixedly connected by the threaded structure.

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

[0204] Specifically, in this embodiment, a first signal transmission component is provided on the nanopore sequencing chip 9, and the first signal transmission component includes a first chip pad 902 and a second electrode line, the first chip pad 902 is provided on the side of the nanopore sequencing chip 9, and the second electrode line is provided inside the nanopore sequencing chip 9, and the second electrode line connects the first chip pad 902 and the electrode 4. The nanopore sequencing chip 9 is provided on the packaging substrate 23.

[0205] Specifically, a second metal lead is also included. A substrate pad 26 is provided on one side of the package substrate 23 where the nanopore sequencing chip 9 is provided, and an array-arranged second metal contact 25 is provided on the other side. The second metal lead is passed through the interior of the package substrate 23, one end of which is connected to the substrate pad 26, and the other end of which is connected to the second metal contact 25. The substrate pad 26 is located on the side of the nanopore sequencing chip 9, and the substrate pad 26 is provided corresponding to the first chip pad 902; one end of the second metal lead connected to the substrate pad 26 extends to the outside of the package substrate 23 and is connected to the first chip pad 902.

[0206] Specifically, an elliptical second through hole 27 is provided inside the polymer gasket 24 , and the second through hole 27 is arranged corresponding to the pore sequencing area 901 on the nanopore sequencing chip 9 .

[0207] Specifically, the bottom shell 22 is placed in the lower layer, and the bottom shell 22 is bonded to the side of the packaging substrate 23 where the second metal contact 25 is set; the side of the packaging substrate 23 where the substrate pad 26 is set is bonded to the polymer gasket 24, and the upper shell 21 covers the surface of the polymer gasket 24 and is fixedly connected to the bottom shell 22.

[0208] Specifically, in this embodiment, the packaging substrate 23 does not include functional modules such as an integrated signal processing circuit, but only includes a simple connection circuit.

[0209] Specifically, in this embodiment, the number of holes 201 , substrate pads 26 , second metal contacts 25 and first chip pads 902 are all 512.

[0210] Example 4

[0211] The difference between this embodiment and embodiment 1 lies in the structure, preparation method and packaging structure of the nanopore sequencing chip. The specific differences are as follows:

[0212] In this embodiment, the height of the oil storage area is 20 μm, and the hydrophobic area further includes a hydrophobic modified area, which is disposed at the top of the hole 201 .

[0213] In this embodiment, the method for preparing the nanopore sequencing chip is different from that in Embodiment 1 in that: after forming the hydrophilic modified region in step S3, the top of the pore 201 is further subjected to hydrophobic modification.

[0214] Specifically, Fig.28 As shown, in this embodiment, the hydrophobic modification is carried out in a closed transparent chamber 20, the top of the chamber 20 is provided with an air inlet 28 and an exhaust port 29, a glue-spreading tray 30 is provided at the center of the bottom, and heating platforms 32 are provided on both sides;

[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 tray 30, and dripping the 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 dripped is 5μL. Heating at a temperature of 100°C for 1min, and then stopping heating; during this period, the glue tray 30 with the hydrophilic modified chip rotates at a uniform speed of 100r / s and high-purity nitrogen is continuously input at the air inlet during the cooling period; the continuous supply of nitrogen can discharge a large amount of fluorine-containing gas and cool the fluorine-containing gas and uniformly deposit it on the surface of the second insulating layer 2 to form a hydrophobic modification area; when the content of the modified reagent is not diluted or nitrogen is not introduced, the deposited hydrophobic film will be too thick, which will lead to failure to conduct or uneven performance during subsequent biological experiments.

[0216] Specifically, the packaging structure of the nanopore sequencing chip in this embodiment is different from that in Embodiment 3 only in that an integrated circuit module 33 and a heat conduction module 34 penetrating the packaging substrate 23 are provided on the packaging substrate 23 of this embodiment. Fig.29 As shown, the integrated circuit module 33 is arranged on the upper and lower surfaces of the packaging substrate 23, and the side surface of the packaging substrate 23 is provided with a data transmission interface 37 composed of a metal connection plug-in, and the signal in the integrated circuit module 33 is transmitted through the data transmission interface 37. A third through hole corresponding to the data transmission interface 37 is provided on the bottom shell 22, and the data transmission interface 37 can be penetrated in the third through hole and extended to the outside of the second plastic package shell.

[0217] Specifically, in this embodiment, there are 384 holes 201 , 384 substrate pads 26 , 384 second metal contacts 25 , and 384 first chip pads 902 .

[0218] Example 5

[0219] Please refer to the following for details Figure 30-33 The difference between this embodiment and embodiment 1 is that:

[0220] In the nanopore sequencing chip of this embodiment, a second signal transmission component is used to replace the first signal transmission component. The second signal transmission component includes 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, and the third electrode line 904 is arranged inside the nanopore sequencing chip 9. The second chip pad 903 is connected to the integrated circuit area 35 through the third electrode line 904.

[0221] Specifically, in this embodiment, the integrated circuit region 35 includes a signal amplifier 3501 , a multiplexer 3502 , and an analog-to-digital converter 3503 which are sequentially connected through a fourth electrode line.

[0222] Specifically, the first insulating layer 1 includes a base layer and an insulating layer 103 , wherein the insulating layer 103 is disposed between the second insulating layer 2 and the base layer; and the integrated circuit region 35 is disposed inside the base layer.

[0223] Specifically, the electrode 4 is disposed on the base layer and communicated with the outside of the first insulating layer 1 , and the first electrode line 36 is disposed inside the base layer and connects the electrode 4 and the integrated circuit region 35 .

[0224] Specifically, a hole sequencing region 901 is formed between the holes 201 arranged in an array on the second insulating layer 2 , and the integrated circuit region 35 is disposed below the hole sequencing region 901 .

[0225] Specifically, the base layer in this embodiment is a single crystal silicon layer 101 , and the integrated circuit region 35 is disposed inside the single crystal silicon layer 101 .

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

[0227] For more details, please refer to Fig.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. After the current signal passes through the signal amplifier 3501, it is transmitted to the multiplexer 3502 through the fourth electrode line. The current signals of multiple electrodes 4 are received simultaneously through a multiplexer 3502, and 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 transmit it to the second chip pad 903 through the third electrode line 904, and output it through the second chip pad 903.

[0228] Specifically, in this embodiment, there are 1920 holes 201 , and there are 96 second chip pads 903 , package frame pads 11 , and first metal contacts 13 .

[0229] In this embodiment, the number of second chip pads 903, package frame pads 11 and first metal contacts 13 does not need to correspond one-to-one to the number of holes 201. Since the multiplexer 3503 can simultaneously receive current signals from multiple electrodes 4, the number of holes 201 can be set to an integer multiple of 96. The current signal can directly convert the real-time measured data through the integrated circuit area 35 and output it through the second chip pad 903, which can reduce the interference generated during signal transmission, while increasing the number and flux of the holes 201 and further reducing the volume.

[0230] Example 6

[0231] The only difference between this embodiment and embodiment 5 is that in this embodiment, there are 1536 holes 201 .

[0232] Example 7

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

[0234] Please refer to the following for details Fig.34 and Fig.35 The difference between the nanopore sequencing chip of this embodiment and that of embodiment 3 is that:

[0235] The nanopore sequencing chip 9 of this embodiment further includes an integrated circuit region 35 and a first electrode line 36 . The integrated circuit region 35 is disposed on the first insulating layer 1 and is connected to the electrode 4 through the first electrode line 36 .

[0236] Specifically, the first insulating layer 1 includes a base layer and an insulating layer 103 , and the insulating layer 103 is disposed between the second insulating layer 2 and the base layer; the integrated circuit region 35 is located between the base layer and the insulating layer 103 .

[0237] Specifically, the electrode 4 is disposed on the base layer and communicated with the outside of the first insulating layer 1 , and the first electrode line 36 is disposed inside the base layer and connects the electrode 4 and the integrated circuit region 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 disposed on the side of the hole sequencing area 901 .

[0239] Specifically, the base layer in this embodiment includes a single crystal silicon layer 101 and a glass layer 102 , the integrated circuit region 35 is disposed 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 103 and the second insulating layer 2 .

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

[0241] Specifically, in this embodiment, there are 1000 holes 201 , and there are 10 second chip pads 903 , 10 substrate pads 26 , and 10 second metal contacts 25 .

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

[0243] Example 8

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

[0245] The first liquid storage tank 15 is composed of two sets of opposing side walls, the lower side wall of the opposing side wall with shorter side length is a vertical structure, the upper side wall is a chamfered design, and the other set of opposing side walls is a vertical structure; the chamfered design is conducive to fluid diffusion and buffering, thereby improving chip performance.

[0246] To illustrate the technical effect of the present invention, the nanopore sequencing chip prepared in Example 1 was subjected to manual pore conduction experiment, membrane laying experiment and biological test experiment. Figures 41-43 As shown, the pore wetting rate of Example 1 is as high as over 97%, the film laying rate and film forming rate are high, and it is easy to use and has high test efficiency, and data can be obtained within 30 minutes.

[0247] In order to further illustrate the technical effect of the present invention, comparative examples 1 and 2 are set up on the basis of embodiment 3.

[0248] The only difference between Comparative Example 1 and Example 3 is that the nanopore sequencing chip is not subjected to hydrophilic modification or hydrophobic modification; the only difference between Comparative Example 2 and Example 3 is that the hydrophilic modification solution is not removed during the hydrophilic modification.

[0249] A water drop test was performed on Example 3, Comparative Example 1 and Comparative Example 2, i.e., pure water was dropped on the top of the hole of the nanopore sequencing chip of Comparative Example 1, Comparative Example 2 and Example 3, and the state of the water drop was observed. The test results are shown in FIG. Figure 38-40 As shown, after pure water is dripped on the top of the pore of the nanopore sequencing chip treated by the method of the present invention, the state of the water drop is more rounded than that of comparative examples 1 and 2, indicating that the treatment scheme of the present invention can significantly improve the hydrophobicity of the top of the pore.

[0250] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A nanopore sequencing chip, characterized in that: The invention comprises 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 holes arranged in an array are arranged on the second insulating layer; the holes penetrate the second insulating layer and are connected with the first insulating layer; the electrodes are arranged at the bottom of the holes; a hydrophobic area is arranged on the second insulating layer, and the hydrophobic area is located above the holes and is arranged around the holes; a hydrophilic modified area is arranged at the bottom of the holes; the hydrophilic modified area is obtained by modifying a hydrophilic modified solution, and the hydrophilic modified solution is selected from a chitosan solution, a dopamine solution, and a polylysine solution.

2. The nanopore sequencing chip according to claim 1, characterized in that: The hydrophobic area is an oil storage area and / or a hydrophobic modified area.

3. The nanopore sequencing chip according to claim 2, characterized in that: The hydrophobic area includes an oil storage area; the oil storage area is arranged on the upper surface of the second insulating layer, and the upper surface of the second insulating layer is provided with a plurality of groove structures or a plurality of boss structures, and the oil storage area is formed between the plurality of groove structures or the plurality of boss structures, and the distance between the oil storage area and the edge of the hole is 3μm-50μm.

4. The nanopore sequencing chip according to claim 3, characterized in that: The upper surface of the second insulating layer is provided with a plurality of boss structures, and the plurality of boss structures form an oil storage area. The shape of the boss structures can be circular, quasi-circular or polygonal; the polygons include regular polygons and irregular polygons.

5. The nanopore sequencing chip according to claim 4, characterized in that: The shape of the boss structure is hexagonal, and a plurality of the hexagonal boss structures are regularly arranged on the second insulating layer; the distance between adjacent hexagonal boss structures is 1 μm-50 μm.

6. The nanopore sequencing chip according to claim 5, characterized in that: Regular flow channels are formed between the plurality of hexagonal boss structures. The regular flow channels and the hexagonal boss structures form a honeycomb structure. The depth / thickness of the regular flow channels is 1 μm-50 μm.

7. The nanopore sequencing chip according to claim 3, characterized in that: The upper surface of the second insulating layer is provided with a plurality of groove structures, the plurality of groove structures form an oil storage area, the shape of the groove structure is circular or polygonal, the plurality of groove structures are regularly arranged to form the oil storage area, and the depth / thickness of the groove structure is 1μm-50μm.

8. The nanopore sequencing chip according to claim 2, wherein: The hydrophobic region includes a hydrophobic modified region, and the hydrophobic modified region is obtained by hydrophobic modification with a hydrophobic modified solution, and the hydrophobic modified solution is selected from one of fluorine-containing compounds, polysilicon compounds, and synthetic carbon chain compounds.

9. The nanopore sequencing chip according to claim 8, wherein: The mass concentration of the hydrophobic modification solution is 10%-60%, and the hydrophobic modification comprises the following steps: The hydrophobic modification solution is dripped onto 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; and then heated at a temperature of 100° C. to 200° C. for 30 min to 180 min to form a hydrophobic area above the hole; The volume of the hydrophobic modified solution added dropwise is 0.5 μL-5 μL.

10. The nanopore sequencing chip according to claim 8, wherein: The hydrophobic modification solution is a fluorine-containing compound, and the hydrophobic modification comprises the following steps: The fluorine-containing compound is heated to be evaporated into gaseous state, and high-purity nitrogen or inert gas is used to drive the gaseous fluorine-containing compound to fill the modified environment and evenly deposit on the surface of the second insulating layer to form a hydrophobic area.

11. The nanopore sequencing chip according to any one of claims 1 to 10, characterized in that: It also includes a signal transmission component, which is connected to the electrode and is used to transmit a test signal.

12. The nanopore sequencing chip according to claim 11, wherein: The signal transmission component comprises an electrode line and a chip pad, wherein the chip pad is arranged outside the first insulating layer or the second insulating layer, and the chip pad is connected to the electrode through the electrode line.

13. The nanopore sequencing chip according to claim 12, wherein: The signal transmission component also includes an integrated circuit area and a first electrode line. The integrated circuit area is arranged on the first insulating layer and is connected to the electrode through the first electrode line. One end of the electrode line is connected to the chip pad and the other end is connected to the integrated circuit area. It is connected to the electrode through the integrated circuit area and the first electrode line; the integrated circuit area includes a signal amplifier, a multiplexer and an analog signal converter; the signal amplifier, the multiplexer and the analog-to-digital converter are connected in sequence through a fourth electrode line.

14. The nanopore sequencing chip according to claim 13, wherein: The first insulating layer includes a base layer and an insulating layer, wherein the insulating layer is arranged between the second insulating layer and the base layer; the integrated circuit area 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 connected with the outside of the first insulating layer; the first electrode line is arranged inside the base layer, connecting the electrode and the integrated circuit area.

15. The nanopore sequencing chip according to claim 14, wherein: The base layer comprises a single crystal silicon layer, and the integrated circuit region is arranged on the single crystal silicon layer.

16. The nanopore sequencing chip according to claim 14, wherein: The base layer also includes a glass layer, and the single crystal silicon layer is located between the glass layer and the insulating layer.

17. The nanopore sequencing chip according to claim 13, wherein: The integrated circuit area is provided with a plurality of them.

18. The nanopore sequencing chip according to any one of claims 12 to 17, wherein: The preparation method of the hydrophilic modified layer comprises the following steps: S1. Hydrophilic modification: Infiltrate the pores with a hydrophilic modification solution having a mass concentration of 5%-20%, and modify at a temperature of 20°C-50°C for 30min-180min; S2, removing the hydrophilic modification solution at the hole mouth and retaining the hydrophilic modification solution at the bottom of the hole, so that the upper end of the hole returns to a hydrophobic state; then aging it for more than 5 days, so that a hydrophilic modification area is formed at the bottom of the hole under the action of the hydrophilic modification solution.

19. The nanopore sequencing chip according to claim 18, wherein: The holes are completely soaked in 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 holes need to be completely discharged so that the inner wall of the hole is completely soaked in the hydrophilic modification solution.

20. A method for preparing a nanopore sequencing chip according to any one of claims 1 to 10, characterized in that: The following steps are involved: Electrodes are arranged on the first insulating layer, and then photoresist is coated on the surface of the first insulating layer to form a photoresist surface. According to the diffraction characteristics of light, a second insulating layer is prepared on the first insulating layer by photolithography exposure technology, so that the pattern on the mask is transferred to the second insulating layer, and a plurality of holes corresponding to the electrodes are formed on the second insulating layer, and an oil storage area is formed at the same time; finally, the bottom of the hole is hydrophilically modified to form a hydrophilic modified area at the bottom to obtain a nanopore sequencing chip.

21. The method for preparing a nanopore sequencing chip according to claim 20, characterized in that: The photoresist surface and the electrode are both located on the same surface of the first insulating layer, and the light-transmitting area of ​​the mask plate making the oil storage area is smaller than or equal to the structural 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, that is, a quasi-double-layer structure.

22. The method for preparing a nanopore sequencing chip according to claim 20, wherein: After the hydrophilic modification treatment, the upper end of the hole and the surface of the second insulating layer are further subjected to hydrophobic modification treatment, thereby forming a hydrophobic modified area on the upper end of the hole and the surface of the second insulating layer.

23. A packaging structure of a nanopore sequencing chip, characterized in that: Comprising the nanopore sequencing chip as described in claim 12.

24. The packaging structure of the nanopore sequencing chip according to claim 23, characterized in that: It also includes a first plastic-sealed shell, a packaging frame and a first metal lead, wherein the first plastic-sealed shell is provided with a first through hole, the packaging frame is installed at the bottom of the first through hole, and the nanopore sequencing chip is stuck inside the packaging frame; a first liquid storage tank is formed between the upper end of the first through hole and the upper surface of the nanopore sequencing chip, and the first liquid storage tank is connected to the holes arranged in the array.

25. The packaging structure of the nanopore sequencing chip according to claim 24, characterized in that: The chip pad is arranged on the side of the nanopore sequencing chip, and the electrode line is arranged inside the nanopore sequencing chip; the side of the packaging frame is provided with a plurality of packaging frame pads corresponding to the chip pad; A plurality of first metal leads are provided corresponding to the chip pads, and one end of each of the first metal leads is connected to the chip pad, and the other end is connected to the package frame pad.

26. The packaging structure of the nanopore sequencing chip according to claim 24, characterized in that: The first through hole can be set as a window, and the shape of the window in top view can be a quadrilateral, a circle, or an irregular shape; the upper surface shape and the lower surface shape of the window can be consistent / or inconsistent; the side wall can be set to be vertical / or non-vertical, or a combination of vertical and non-vertical alternation; The window can be configured as a cuboid, a through hole, an inverted frustum, or an irregular groove.

27. The packaging structure of the nanopore sequencing chip according to claim 24, characterized in that: A plurality of first metal contacts corresponding to the packaging frame pads are arranged around the outer side of the bottom of the plastic package shell, and the first metal contacts are electrically connected to the packaging frame pads.

28. The packaging structure of the nanopore sequencing chip according to claim 23, characterized in that: It also includes a second plastic packaging shell, a packaging substrate, a polymer gasket and a second metal lead; the nanopore sequencing chip is arranged on the packaging substrate, the packaging substrate is arranged inside the second plastic packaging shell, and the polymer gasket is arranged between the nanopore sequencing chip and the second plastic packaging shell.

29. The packaging structure of the nanopore sequencing chip according to claim 28, characterized in that: The chip pad is arranged on the side of the nanopore sequencing chip, and the electrode line is arranged inside the nanopore sequencing chip; the packaging substrate is provided with a substrate pad corresponding to the chip pad on one side of the nanopore sequencing chip, and an array-arranged second metal contact is arranged on the other side; the second metal lead is passed through the interior of the packaging substrate, one end of which is connected to the substrate pad, and the other end of which is connected to the second metal contact; one end of the second metal lead connected to the substrate pad extends to the outside of the packaging substrate and is connected to 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 holes arranged in an array on the nanopore sequencing chip.

30. The packaging structure of the nanopore sequencing chip according to claim 29, characterized in that: The second plastic-sealed housing comprises an upper housing and a bottom housing, the nanopore sequencing chip is located between the upper housing and the bottom housing, and the front side of the nanopore sequencing chip is arranged toward the upper housing; 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 to connect the second liquid storage tank and the waste liquid tank. The valve is located at the top of the upper shell. The valve is used to control whether the flow channel is connected. The bottom shell is provided with a base card slot, and the package base card is arranged in the base card slot; The upper shell and the bottom shell are detachably connected.

31. The packaging structure of the nanopore sequencing chip according to any one of claims 28 to 30, characterized in that: The packaging substrate may also be provided with an integrated circuit module and a heat-conducting module that passes through the packaging substrate, the integrated circuit module is arranged on the side of the heat-conducting module, and the nanopore sequencing chip is arranged on the heat-conducting module; a data transmission interface is provided on the side of the packaging substrate, and a third through hole corresponding to the data transmission interface is provided on the bottom shell, and the data transmission interface can be passed through the third through hole and extend to the outside of the second plastic packaging shell.

Images