Nanopore sequencing pretreatment chip as well as preparation method and application thereof

Abstract

The invention discloses a nanopore sequencing pretreatment chip as well as a preparation method and application thereof, and belongs to the technical field of microfluidics. The invention provides a nanopore sequencing pretreatment chip, the chip is composed of a conductive glass layer, a reaction liquid layer and a printed circuit board layer, and the reaction liquid layer comprises a nucleic acid extraction unit, a gene library construction unit and a nucleic acid concentration determination unit. The invention further provides a preparation method based on the chip and application of the chip in sequencing sample preparation. According to the invention, high integration of core steps of sequencing pretreatment is realized, the preparation efficiency is improved, an effective scheme is provided for full automation of sequencing pretreatment, and the method can be widely applied to high-throughput and ultra-sensitive sequencing pretreatment of clinical samples.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics technology, and specifically relates to a nanopore sequencing pretreatment chip, its preparation method, and its application. Background Technology

[0002] The pretreatment steps for nanopore sequencing typically include sample nucleic acid extraction, gene library construction, and DNA concentration determination. Currently available instruments and microfluidic chips can only automate some of these processes. Multiple instruments, such as centrifuges, PCR machines, vortex mixers, and qubit quantitative PCR instruments, are still needed to complete steps like sample mixing and DNA concentration determination. Furthermore, DNA concentration determination relies on external equipment, resulting in data latency >10 minutes. In addition, the sequencing sample preparation process is complex, typically requiring 4-6 hours and involving numerous steps such as pipetting, washing, and incubation, demanding high levels of expertise. Therefore, developing highly integrated and fully automated pretreatment chips and instruments is a pressing technical challenge that needs to be addressed. Summary of the Invention

[0003] Based on the technical problems to be solved by the present invention, the present invention proposes a nanopore sequencing pretreatment chip composed of a three-layer structure of a conductive glass layer, a reaction liquid layer and a printed circuit board layer, and further proposes a method for preparing the nanopore sequencing pretreatment chip and a sequencing sample preparation application based on the nanopore sequencing pretreatment chip.

[0004] One of the objectives of this invention is to provide a nanopore sequencing pretreatment chip, which includes a conductive glass layer, a reaction liquid layer, and a printed circuit board (PCB) layer. The conductive glass layer includes a hydrophobic layer and a sample application port; the conductive glass layer is made of light-transmitting glass and is used to collect fluorescence signals. The reaction liquid layer is provided with a nucleic acid extraction unit, a gene library construction unit, a nucleic acid concentration measurement unit, and a paraffin valve for measuring the nucleic acid extraction concentration and the gene library concentration; The nucleic acid extraction unit includes a lysis chamber 8, a lysis washing chamber 6, and an extraction elution chamber 3, used for extracting and purifying nucleic acid samples; isolation channels are provided between the chambers. The gene library construction unit includes a nucleic acid repair chamber 4, two repair washing chambers, a repair elution chamber 9, a adapter ligation chamber 11, a ligation washing chamber 12, a gene library elution chamber 13, and two magnetic bead pre-storage chambers, used for nucleic acid end repair, washing purification, and adapter ligation; isolation channels are provided between the chambers; The nucleic acid concentration determination unit includes two nucleic acid dilution chambers, two fluorescent dye chambers, and two nucleic acid concentration determination chambers, used for quantitative detection of nucleic acid products in the extraction and library construction stages, respectively; isolation channels are provided between the chambers; The paraffin valve is used to regulate the opening and closing state of the isolation channel; The printed circuit board layer, corresponding to the nucleic acid dilution chamber, the fluorescent dye chamber, the nucleic acid concentration measurement chamber, the repair elution chamber, and the magnetic bead pre-storage chamber, and one side surface of its connecting channel is provided with array electrodes to provide power to the reaction liquid layer; the surface of the printed circuit board layer is provided with a heating resistance wire corresponding to the bottom of the paraffin valve to melt the paraffin valve.

[0005] Furthermore, the hydrophobic layer is disposed on the surface of the conductive glass layer. The hydrophobic coating solution is dropped onto the center of the clean glass surface activated by plasma. The centrifugal force generated by the high-speed rotation of the spin coater causes the droplet to spread into a uniform thin layer within a few seconds and remove excess liquid. Subsequently, medium-temperature pre-curing is performed to allow the solvent to evaporate steadily. Then, it is transferred to a high-temperature oven to promote the cross-linking reaction between the hydrophobic material and the silanol groups on the glass surface, ultimately forming a strong, smooth, and highly hydrophobic covalently bonded coating, which is used to control the surface hydrophilicity and hydrophobicity of the reaction liquid layer.

[0006] Furthermore, there are 16 sample loading wells, which are respectively set on the conductive glass layer surface corresponding to the 16 functional chambers other than the nucleic acid concentration determination chamber, for adding solution to the 16 functional chambers.

[0007] Further, the two nucleic acid dilution chambers include nucleic acid dilution chamber 2 and nucleic acid dilution chamber 14; nucleic acid dilution chamber 2 is used to add enzyme-free sterile water or nucleic acid solution; nucleic acid dilution chamber 14 is used to add enzyme-free sterile water or gene library solution; extraction elution chamber 3 adds extraction elution buffer; nucleic acid repair chamber 4 adds nucleic acid repair solution; the two repair washing chambers include repair washing chamber 5 and repair washing chamber 7, both used to add repair washing solution; lysis washing chamber 6 adds lysis washing solution; lysis chamber 8 adds lysis mixture or sample solution; repair elution chamber 9 adds repair elution solution; the two magnetic bead pre-storage chambers include... The magnetic bead pre-storage chambers 10 and 15 are both used for adding purification magnetic beads; the adapter connection chamber 11 is used to add adapter connection solution; the connection washing chamber 12 is used to add connection washing solution; the gene library elution chamber 13 is used to add gene library elution solution; the two fluorescent dye chambers, including fluorescent dye chamber 17 and fluorescent dye chamber 18, are both used for adding fluorescent dye; the two nucleic acid concentration measurement chambers, including nucleic acid concentration measurement chamber 1 and nucleic acid concentration measurement chamber 16, are used to store nucleic acid mixed solution transferred in by electrode drive; nucleic acid concentration measurement chamber 16 is used to store gene library mixed solution transferred in by electrode drive. Furthermore, the third, fifth, and sixth isolation channels are connected to form a nucleic acid solution "dilution-mixing-testing" channel; the fifteenth, sixteenth, and seventeenth isolation channels are connected to form a gene library solution "dilution-mixing-testing" channel; the two "dilution-mixing-testing" channels are independent of each other; A second objective of this invention is to provide a method for preparing a nanopore sequencing pretreatment chip, the method comprising: S1, add enzyme-free sterile water to the nucleic acid dilution chamber 2 and the nucleic acid dilution chamber 14, add extraction elution buffer to the extraction elution chamber 3, add nucleic acid repair buffer to the nucleic acid repair chamber 4, add repair washing buffer to the repair washing chamber 5 and the repair washing chamber 7, add lysis washing buffer to the lysis washing chamber 6, add lysis mixture to the lysis chamber 8, add repair elution buffer to the repair elution chamber 9, add purified magnetic beads to the magnetic bead pre-storage chamber 10 and the magnetic bead pre-storage chamber 15, add adapter ligation solution to the adapter ligation chamber 11, add ligation washing solution to the ligation washing chamber 12, add gene library elution solution to the gene library elution chamber 13, add fluorescent dye to the fluorescent dye chamber 17 and the fluorescent dye chamber 18, and add sample solution to the lysis chamber 8; S2, incubate the lysis chamber 8 at 70~80℃ for 10~20min; use an external magnet to drive the magnetic beads in the lysis chamber 8 through the lysis washing chamber 6 and the extraction elution chamber 3 in sequence, and elute at 50~60℃ for 2~15min to obtain a nucleic acid solution; S3, the nucleic acid solution is transferred to the nucleic acid dilution chamber 2 for dilution, and then transferred to the fluorescent dye chamber 18 for mixing. It is then incubated at room temperature in the dark for 1-3 minutes to obtain a nucleic acid mixture. S4, the nucleic acid mixture is transferred to the nucleic acid concentration determination chamber 1 for initial quantitative detection, and the nucleic acid concentration is calculated; S5, transfer the nucleic acid mixture to the nucleic acid repair chamber 4, incubate at 15~25℃ for 1~10 min, incubate at 60~70℃ for 1~10 min, mix thoroughly to obtain the nucleic acid end repair mixture; S6, drive the magnetic beads in the magnetic bead pre-storage chamber 15 to move to the nucleic acid repair chamber 4, mix with the nucleic acid end repair mixture, and incubate at room temperature for 5-15 min; drive the magnetic beads to pass through the repair washing chambers 5 and 7 in sequence to mix thoroughly; drive the magnetic beads to the repair elution chamber 9, incubate at room temperature for 1-3 min, and obtain the purified and eluted nucleic acid solution. S7. Using electrode drive, the purified and eluted nucleic acid solution is moved to the adapter connection chamber 11, thoroughly mixed, and incubated at room temperature for 5-15 min; the magnetic beads in the magnetic bead pre-storage chamber 10 are moved to the adapter connection chamber 11, thoroughly mixed, and incubated at room temperature for 5-15 min; the magnetic beads are moved to the connection washing chamber 12, thoroughly mixed; the magnetic beads are moved to the gene library elution chamber 13, thoroughly mixed, and incubated at room temperature for 5-15 min to obtain the gene library elution buffer; S8, the gene library elution buffer is transferred to the nucleic acid dilution chamber 14 for dilution to obtain a nucleic acid diluent; the nucleic acid diluent is transferred to the fluorescent dye chamber 17, thoroughly mixed, and incubated at room temperature in the dark for 1-3 minutes to obtain a gene library mixture; S9, the gene library mixture is transferred to the nucleic acid concentration determination chamber 16, and the gene library concentration is determined.

[0008] Furthermore, the nucleic acid solution is diluted in the nucleic acid dilution chamber 2 at a volume ratio of 1:2 to 10; the gene library solution is diluted in the nucleic acid dilution chamber 14 at a volume ratio of 1:2 to 10.

[0009] Furthermore, the gene library solution can be directly used for sequencing.

[0010] The third objective of this invention is to provide an application of a nanopore sequencing pretreatment chip, or a nanopore sequencing pretreatment chip prepared by a nanopore sequencing pretreatment chip preparation method, in the sequencing sample preparation of blood, saliva, or bacterial culture medium.

[0011] Compared with existing technologies, this invention proposes a nanopore sequencing pretreatment chip, which has the following advantages: This invention provides a nanopore sequencing pretreatment chip that simultaneously performs sample nucleic acid extraction, library construction, and DNA concentration determination, significantly improving sample preparation efficiency and providing a foundation for automated control of nanopore sequencing sample pretreatment preparation.

[0012] Furthermore, the conductive glass layer of the chip in this invention is made of light-transmitting glass to achieve fluorescence signal acquisition. The sample application holes on the conductive glass layer are physically separated by paraffin valves to achieve precise control of reagent storage.

[0013] Furthermore, the reaction liquid layer of the chip in this invention adopts a single-sample, single-channel design, while the functional chambers are connected in series through isolation channels, forming two independent "dilution-mixing-determination" paths. This enables high-throughput automated preparation of nucleic acid products at different stages without cross-contamination and for various sample types. In the fluorescent dye chamber within the "dilution-mixing-determination" path, the volume of fluorescent dye used is optimized, reducing the volume of fluorescent dye used and achieving rapid mixing and integration.

[0014] Furthermore, the printed circuit board (PCB) layer is equipped with array electrodes in key areas such as the nucleic acid dilution chamber, the fluorescent dye chamber, the nucleic acid concentration measurement chamber, the repair elution chamber, and the magnetic bead pre-storage chamber, as well as on one side of the connecting channel, which significantly reduces the chip fabrication cost. Attached Figure Description

[0015] Figure 1 This is a flowchart for chip usage.

[0016] Figure 2 This diagram illustrates the chip structure design and chamber functions; where 1-nucleic acid concentration measurement chamber; 2-nucleic acid dilution chamber; 3-extraction and elution chamber; 4-nucleic acid repair chamber; 5-repair and washing chamber; 6-lysis and washing chamber; 7-repair and washing chamber; 8-lysis chamber; 9-repair and elution chamber; 10-magnetic bead pre-storage chamber; 11-connector ligation chamber; 12-ligation and washing chamber; 13-gene library elution chamber; 14-nucleic acid dilution chamber; 15-magnetic bead pre-storage chamber; 16-nucleic acid concentration measurement chamber; 17-fluorescent dye chamber; and 18-fluorescent dye chamber. Detailed Implementation

[0017] Exemplary embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present application are shown in the drawings, it should be understood that the present application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0018] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the reagents and materials in this invention are obtained from the market or other public channels.

[0019] Example 1 This invention proposes a nanopore sequencing pretreatment chip.

[0020] The chip of this invention adopts a three-layer stacked design, such as... Figure 2 As shown, it includes an upper conductive glass layer (500μm thick), an intermediate reactive liquid layer (800μm thick), and a lower PCB layer (700μm thick), with overall dimensions of 20mm × 150mm × 3mm (length × width × height). The structural parameters and functional coordination of each layer are as follows: The upper layer uses borosilicate conductive and light-transmitting glass with a transmittance of ≥90% in the 400-700nm wavelength range. A hydrophobic coating solution is dropped onto the center of the glass surface that has been plasma-deposited and cured. Centrifugal force generated by the high-speed rotation of a spin coater causes the solution droplet to spread into a uniform thin layer within seconds and removes excess liquid. After medium-temperature pre-curing to allow the solvent to evaporate smoothly, it is then transferred to a high-temperature oven to promote the cross-linking reaction between the hydrophobic material and the silanol groups on the glass surface, forming a strong, smooth, and highly hydrophobic covalently bonded coating with a thickness of 50nm. Sixteen circular sample loading holes with a diameter of 2mm and a depth of 500μm are made at the corresponding 16 functional chambers of the middle layer. The hole walls are silanized with 3-aminopropyltriethoxysilane to avoid reagent residue.

[0021] The intermediate layer comprises 18 functional chambers: lysis chamber 8 (500 μL); repair washing chambers 5, lysis washing chambers 6, lysis washing chambers 7, and connection washing chamber 12 (200 μL each); extraction elution chambers 3, repair elution chambers 9, and gene library elution chamber 13 (100 μL each); nucleic acid dilution chambers 2 and 14 (100 μL each); fluorescent dye chambers 17 and 18 (50 μL each); nucleic acid concentration assay chambers 1 and 16 (10 μL each); and magnetic bead pre-storage chambers 10 and 15 (50 μL each). The functional chambers are connected by isolation channels with a width of 200 μm and a depth of 100 μm. The channel is embedded with a paraffin valve containing medical-grade solid paraffin or filled with a mixed oil, which contains at least one or more of silicone oil, alkane oil, mineral oil, fluorinated oil, and surfactants to achieve liquid isolation and directional flow between chambers. The specific connection relationship is as follows: functional chambers 8 and 6 are connected, 6 and 3 are connected, 3 is connected to 2 and 4 respectively, 2 is connected to 18, 18 is connected to 1, 4 is connected to 15 and 5 respectively, 5 is connected to 7, 7 is connected to 9, 9 is connected to 11, 11 is connected to 10 and 12 respectively, 12 is connected to 13, 13 is connected to 14, 14 is connected to 17, 17 is connected to 16, while 1 and 16 are adjacent but not connected.

[0022] The lower layer uses FR-4 epoxy resin substrate material; ITO transparent electrode arrays are arranged in the corresponding functional chambers 1, 9, 10, 15, 16 and the connecting channel area, with an electrode width of 100μm, a spacing of 50μm, and an operating voltage of 10-30V DC; a nickel-chromium alloy resistance wire (50μm in diameter, 10Ω resistance) with a rated power of 0.5W is embedded at the bottom of the corresponding middle layer paraffin valve, which can raise the temperature of the paraffin valve to above 50℃ and melt it within 3 seconds after being energized; except for the working area of ​​the electrodes and resistance wire, the remaining areas are coated with a 50μm thick epoxy resin insulating layer.

[0023] Example 2 This invention proposes a method for preparing a nanopore sequencing pretreatment chip and its application.

[0024] 1. Sample pretreatment: The overnight culture of the domestic standard strain Pseudomonas aeruginosa CICC 10299 was selected as the sample. 1 mL of the culture was placed in a sterile centrifuge tube and centrifuged at 10,000 rpm for 2 minutes. The supernatant was removed, and the bottom precipitate was retained. 1 mL of PBS buffer was added to the precipitate, and the centrifuge tube was placed on a vortex mixer and vortexed at a medium-low speed of 1,000-1,500 rpm for 30 seconds to initially disperse the precipitate. Then, the PBS buffer in the tube was aspirated with a sterile pipette, and the bottom precipitate was repeatedly pipetted 5-8 times to ensure that the bacteria were completely detached from the tube wall and evenly dispersed in the buffer solution, thus obtaining the pretreated Pseudomonas aeruginosa sample solution.

[0025] 2. Sources and components of reagents used in the chip reaction (see Table 1) All reagents used in this embodiment are commercial standard reagent kits, and the specific models and manufacturers are as follows: Table 1 List of Main Reagents 3. Sample pretreatment method for Pseudomonas aeruginosa nanopore sequencing (1) Reagent and sample loading: Add 5 μl of enzyme-free sterile water to chambers 2 and 14 respectively; add 20 μl of extraction elution buffer to chamber 3; add 6 μl of nucleic acid repair buffer to chamber 4; add 60 μl of repair washing buffer to chamber 5; add 100 μl of lysis washing buffer to chamber 6; add 60 μl of repair washing buffer to chamber 7; and add 187 μl of lysis mixture (containing 60 μl of tissue digestion solution, 60 μl of lysis buffer, 60 μl of isopropanol, and 3 μl of purification magnetic beads) to chamber 8. Add 4 μl of proteinase K to chamber 9, 30 μl of repair elution buffer to chambers 10 and 15 respectively, 20 μl of purification magnetic beads to chamber 11, 20 μl of adapter ligation buffer to chamber 12, 60 μl of ligation washing buffer to chamber 13, 15 μl of gene library elution buffer to chamber 13, and 30 μl of fluorescent dye to chambers 17 and 18 respectively. Add 150 μl of pretreated Pseudomonas aeruginosa sample solution to lysis chamber 8 of the chip to begin the nanopore sequencing pretreatment preparation.

[0026] (2) Nucleic acid extraction: The chip is placed in the slot of the electrode control system. The No. 8 chamber is set to incubate at 75°C for 15 minutes. During this period, the chip is moved clockwise every 3 minutes by an external magnet (magnetic strength 100mT) for 10 seconds each time to achieve thorough mixing of the magnetic beads with the lysis buffer and sample solution and thorough lysis of the nucleic acid in the sample. The magnetic beads are driven to the No. 6 washing chamber and thoroughly mixed with the washing buffer by the action of the magnetic rod. The magnetic beads are driven to the No. 3 elution chamber and thoroughly mixed with the elution buffer. The chip is incubated at 56°C for 10 minutes for elution. During this period, the chip is mixed for 10 seconds every 2 minutes (frequency 50Hz) to promote the elution of nucleic acid from the magnetic beads.

[0027] (3) Nucleic acid concentration determination: Transfer 1 μl of the extraction eluent from elution chamber 3 to nucleic acid dilution chamber 2, shake for 1 minute to mix thoroughly, achieving a 1:6 dilution. Then transfer 1 μl of the dilution to fluorescent dye chamber 18, mix thoroughly, and incubate at room temperature in the dark for 2 minutes; then transfer 5 μl of the mixture to nucleic acid concentration determination chamber 1, start the optical detection equipment, set the excitation wavelength to (460±20) nm and the emission wavelength to (525±20) nm, and collect the fluorescence signal intensity; draw a preset standard curve (using dsDNA standards of known concentrations) to obtain the concentration of the standard. The concentration of extracted nucleic acid was obtained by converting ≥0.995, thus completing the initial quantitative detection of nucleic acid concentration.

[0028] (4) Nucleic acid end repair: Calculate the volume of nucleic acid required to construct the gene library based on the initial concentration test results. Move 1000 ng (16.6 μl) of the extracted nucleic acid to the No. 4 nucleic acid repair chamber and mix thoroughly. Incubate at 20°C for 5 minutes and at 65°C for 5 minutes to complete the end repair reaction of nucleic acid. Drive the magnetic beads in the No. 15 chamber to the No. 4 nucleic acid repair chamber and mix them with the end repair mixture containing the added nucleic acid. Incubate at room temperature for 10 minutes. Drive the magnetic beads through the No. 5 repair washing chamber and the No. 7 repair washing chamber in sequence and mix them thoroughly with the washing solution. Drive the magnetic beads to the No. 9 repair elution chamber and mix them thoroughly with the elution solution. Incubate at room temperature for 2 minutes to achieve the purification and elution of the repaired nucleic acid, and obtain the purified repaired nucleic acid solution, i.e., the supernatant.

[0029] (5) Adapter ligation: Drive the electrode to move all the liquid in the repair elution chamber 9 to the adapter ligation chamber 11 and mix thoroughly. Incubate at room temperature for 10 minutes to complete the adapter ligation reaction of nucleic acid. Drive the magnetic beads in chamber 10 to move to the adapter ligation chamber 11 and mix thoroughly. Incubate at room temperature for 10 minutes. Drive the magnetic beads to the ligation washing chamber 12 and mix thoroughly. Drive the magnetic beads to the gene library elution chamber 13 and mix thoroughly with the elution buffer. Incubate at room temperature for 10 minutes to achieve purification and elution of nucleic acid after ligation.

[0030] (6) Gene library concentration determination: Transfer 1 μl of gene library elution buffer to nucleic acid dilution chamber 14, mix with enzyme-free sterile water by shaking to achieve a 1:6 dilution; transfer 1 μl of dilution buffer to fluorescent dye chamber 17, mix thoroughly, and incubate at room temperature in the dark for 2 minutes; transfer 5 μl of the mixture to nucleic acid concentration determination chamber 16, collect fluorescence signals using optical detection equipment, and complete the gene library concentration determination using the same method as the nucleic acid extraction concentration determination. Calculate the gene library concentration based on the standard curve. The overall process is as follows: Figure 1 As shown.

[0031] result: The results of the two nucleic acid concentration measurements are shown in the table below: Table 2. List of results from two nucleic acid concentration measurements Example 3 This invention proposes a method for preparing a nanopore sequencing pretreatment chip and its application.

[0032] Select clinical peripheral blood samples containing EDTA anticoagulation, and complete sample pretreatment and reagent loading according to the steps and reagents in the aforementioned embodiments. The difference is that 200 μL of pretreated clinical blood sample solution was added to chamber 8. Nucleic acid extraction: Red blood cells were lysed by incubation at 37°C for 10 minutes in chamber 8, followed by incubation at 75°C for 15 minutes to lyse white blood cells, with the magnetic beads driven to mix for 10 seconds every 3 minutes during this period; the magnetic beads were then washed in chamber 6 and mixed for 30 seconds before being transferred to chamber 3 and incubated at 56°C for 10 minutes to elute nucleic acids.

[0033] Complete the extraction of nucleic acid concentration, nucleic acid end repair, adapter ligation, and library concentration determination according to the steps in the aforementioned embodiments.

[0034] result: The results of the two nucleic acid concentration measurements are shown in the table below: Table 3. List of results from two nucleic acid concentration measurements It should be noted that the term "comprising," or any other variation thereof, is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0035] The above are merely embodiments of this application and are not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A nanopore sequencing pretreatment chip, characterized in that, It includes a conductive glass layer, a reactive liquid layer, and a printed circuit board layer; The conductive glass layer includes a hydrophobic layer and a sample application port; the conductive glass layer is made of light-transmitting glass and is used to collect fluorescence signals. The reaction liquid layer is provided with a nucleic acid extraction unit, a gene library construction unit, a nucleic acid concentration measurement unit, and a paraffin valve for measuring the nucleic acid extraction concentration and the gene library concentration; The nucleic acid extraction unit includes a lysis chamber (8), a lysis washing chamber (6), and an extraction elution chamber (3), used for extracting and purifying nucleic acid samples; isolation channels are provided between the chambers; The gene library construction unit includes a nucleic acid repair chamber (4), two repair washing chambers, a repair elution chamber (9), a adapter ligation chamber (11), a ligation washing chamber (12), a gene library elution chamber (13), and two magnetic bead pre-storage chambers, used for nucleic acid end repair, washing purification, and adapter ligation; isolation channels are provided between the chambers; The nucleic acid concentration determination unit includes two nucleic acid dilution chambers, two fluorescent dye chambers, and two nucleic acid concentration determination chambers, used for quantitative detection of nucleic acid products in the extraction and library construction stages, respectively; isolation channels are provided between the chambers; The paraffin valve is used to regulate the opening and closing state of the isolation channel; The printed circuit board layer, corresponding to the nucleic acid dilution chamber, the fluorescent dye chamber, the nucleic acid concentration measurement chamber, the repair elution chamber, and the magnetic bead pre-storage chamber, and one side surface of its connecting channel is provided with array electrodes to provide power to the reaction liquid layer; the surface of the printed circuit board layer is provided with a heating resistance wire corresponding to the bottom of the paraffin valve to melt the paraffin valve.

2. The nanopore sequencing pretreatment chip according to claim 1, characterized in that, The hydrophobic layer is disposed on the surface of the conductive glass layer, and the hydrophobic layer is cross-linked with the silanol groups on the surface of the conductive glass layer to form covalent bonds and a coating, which is used to regulate the surface hydrophilicity and hydrophobicity of the reactive liquid layer.

3. The nanopore sequencing pretreatment chip according to claim 1, characterized in that, The number of sample application wells is 16, which are respectively set on the conductive glass layer surface of the 16 functional chambers other than the nucleic acid concentration determination chamber, and are used to add solution to the 16 functional chambers.

4. The nanopore sequencing pretreatment chip according to claim 1, characterized in that, The nucleic acid dilution chambers are two in number, including a nucleic acid dilution chamber (2) and a nucleic acid dilution chamber (14); the nucleic acid dilution chamber (2) is used to add enzyme-free sterile water or nucleic acid solution; the nucleic acid dilution chamber (14) is used to add enzyme-free sterile water or gene library solution. The extraction and elution chamber (3) is used to add the extraction and elution solution; The nucleic acid repair chamber (4) is used to add nucleic acid repair solution; The two repair washing chambers include a repair washing chamber (5) and a repair washing chamber (7), both of which are used to add repair washing solution; The pyrolysis washing chamber (6) is used to add pyrolysis washing solution; The lysis chamber (8) is used to add lysis mixture or sample solution; The repair elution chamber (9) is used to add repair elution solution; The magnetic bead pre-storage chambers are two in number, including a magnetic bead pre-storage chamber (10) and a magnetic bead pre-storage chamber (15), both of which are used to add purified magnetic beads; The connector connection chamber (11) is used to add connector connection fluid; The connecting washing chamber (12) is used to add connecting washing fluid; The gene library elution chamber (13) is used to add gene library elution buffer; The fluorescent dye chambers are two in number, including a fluorescent dye chamber (17) and a fluorescent dye chamber (18), both of which are used to add fluorescent dye; The nucleic acid concentration measurement chamber consists of two chambers, including a nucleic acid concentration measurement chamber (1) and a nucleic acid concentration measurement chamber (16). The nucleic acid concentration measurement chamber (1) is used to store the extracted nucleic acid mixture solution that is moved in by the electrode. The nucleic acid concentration measurement chamber (16) is used to store the gene library mixture solution that is moved in by the electrode.

5. The nanopore sequencing pretreatment chip according to claim 1, characterized in that, The lysis chamber (8) is connected to the lysis washing chamber (6) via a first isolation channel; the lysis washing chamber (6) is connected to the extraction elution chamber (3) via a second isolation channel; the extraction elution chamber (3) is connected to the nucleic acid dilution chamber (2) via a third isolation channel, and to the nucleic acid repair chamber (4) via a fourth isolation channel; the nucleic acid dilution chamber (2) is connected to the fluorescent dye chamber (18) via a fifth isolation channel; the fluorescent dye chamber (18) is connected to the nucleic acid concentration determination chamber (1) via a sixth isolation channel. The nucleic acid repair chamber (4) is connected to the magnetic bead pre-storage chamber (15) through a seventh isolation channel and to the repair washing chamber (5) through an eighth isolation channel; the repair washing chamber (5) is connected to the repair washing chamber (7) through a ninth isolation channel; the repair washing chamber (7) is connected to the repair elution chamber (9) through a tenth isolation channel; the repair elution chamber (9) is connected to the connector connection chamber (11) through an eleventh isolation channel. The connector connection chamber (11) is connected to the magnetic bead pre-storage chamber (10) via the twelfth isolation channel, and to the connection washing chamber (12) via the thirteenth isolation channel; the connection washing chamber (12) is connected to the gene library elution chamber (13) via the fourteenth isolation channel; the gene library elution chamber (13) is connected to the nucleic acid dilution chamber (14) via the fifteenth isolation channel; the nucleic acid dilution chamber (14) is connected to the fluorescent dye chamber (17) via the sixteenth isolation channel; the fluorescent dye chamber (17) is connected to the nucleic acid concentration determination chamber (16) via the seventeenth isolation channel. The nucleic acid concentration measurement chamber (1) and the nucleic acid concentration measurement chamber (16) are adjacent but not connected.

6. The nanopore sequencing pretreatment chip according to claim 5, characterized in that, The third, fifth, and sixth isolation channels are connected to form a nucleic acid solution "dilution-mixing-determination" channel; The fifteenth isolation channel, the sixteenth isolation channel, and the seventeenth isolation channel are connected to form a gene library solution "dilution-mixing-determination" channel; The two "dilution-mixing-determination" channels are independent of each other.

7. A method for preparing a nanopore sequencing pretreatment chip, characterized in that, The method, applied to the nanopore sequencing pretreatment chip according to claims 1-6, comprises: S1, add enzyme-free sterile water to the nucleic acid dilution chamber (2) and the nucleic acid dilution chamber (14), add extraction elution buffer to the extraction elution chamber (3), add nucleic acid repair solution to the nucleic acid repair chamber (4), add repair washing solution to the repair washing chamber (5) and the repair washing chamber (7), add lysis washing solution to the lysis washing chamber (6), add lysis mixture to the lysis chamber (8), add repair elution solution to the repair elution chamber (9), add purification magnetic beads to the magnetic bead pre-storage chamber (10) and the magnetic bead pre-storage chamber (15), add adapter connection solution to the adapter connection chamber (11), add connection washing solution to the connection washing chamber (12), add gene library elution solution to the gene library elution chamber (13), add fluorescent dye to the fluorescent dye chamber (17) and the fluorescent dye chamber (18), and add sample solution to the lysis chamber (8); S2, incubate the lysis chamber (8) at 70~80℃ for 10~20min; drive the magnetic beads in the lysis chamber (8) with an external magnet, and pass them sequentially through the lysis washing chamber (6) and the extraction elution chamber (3), elute at 50~60℃ for 2~15min to obtain a nucleic acid solution; S3, the nucleic acid solution is transferred to the nucleic acid dilution chamber (2) for dilution, and then transferred to the fluorescent dye chamber (18) for mixing. It is then incubated at room temperature in the dark for 1-3 minutes to obtain a nucleic acid mixture. S4, the nucleic acid mixture is transferred to the nucleic acid concentration determination chamber (1) for initial quantitative detection, and the nucleic acid concentration is calculated; S5, transfer the nucleic acid mixture to the nucleic acid repair chamber (4), incubate at 15~25℃ for 1~10 min, incubate at 60~70℃ for 1~10 min, mix thoroughly to obtain nucleic acid end repair mixture; S6, drive the magnetic beads in the magnetic bead pre-storage chamber (15) to move to the nucleic acid repair chamber (4), mix with the nucleic acid end repair mixture, and incubate at room temperature for 5-15 min; drive the magnetic beads through the repair washing chamber (5) and the repair washing chamber (7) in sequence, mix thoroughly; drive the magnetic beads to the repair elution chamber (9), incubate at room temperature for 1-3 min, and obtain the purified and eluted nucleic acid solution; S7. Using electrode drive, the purified and eluted nucleic acid solution is moved to the adapter connection chamber (11), thoroughly mixed, and incubated at room temperature for 5-15 min; the magnetic beads in the magnetic bead pre-storage chamber (10) are moved to the adapter connection chamber (11), thoroughly mixed, and incubated at room temperature for 5-15 min; the magnetic beads are driven to the connection washing chamber (12), thoroughly mixed; the magnetic beads are driven to the gene library elution chamber (13), thoroughly mixed, and incubated at room temperature for 5-15 min to obtain the gene library elution buffer; S8, the gene library elution buffer is transferred to the nucleic acid dilution chamber (14) for dilution to obtain a nucleic acid diluent; the nucleic acid diluent is transferred to the fluorescent dye chamber (17), mixed thoroughly, and incubated at room temperature in the dark for 1-3 minutes to obtain a gene library mixture; S9, the gene library mixture is transferred to the nucleic acid concentration determination chamber (16) to determine the gene library concentration.

8. The method for preparing the nanopore sequencing pretreatment chip according to claim 7, characterized in that, The nucleic acid solution is diluted in the nucleic acid dilution chamber (2) at a volume ratio of 1:2 to 10; the gene library solution is diluted in the nucleic acid dilution chamber (14) at a volume ratio of 1:2 to 10.

9. The method for preparing the nanopore sequencing pretreatment chip according to claim 7, characterized in that, The gene library solution is used directly for sequencing.

10. The application of a nanopore sequencing pretreatment chip according to any one of claims 1 to 6, or a nanopore sequencing pretreatment chip prepared by the preparation method according to any one of claims 7 to 9, in the sequencing sample preparation of blood, saliva or bacterial culture medium.

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